Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a Schematic of the screening workflow to identify the E3 ubiquitin ligase FBXO7 as a protein candidate downregulating PRMT1. b Venn diagram showing overlap of proteins among four datasets obtained from BioGRID, GSEA-MSIGDB, MGI, and TCGA. c , d Reciprocal co-IP analysis of PRMT1 and FBXO7 in Huh7 and PLC/PRF/5 cells. IgG was used as a negative control. The immunoblotting experiments were repeated three times with similar results. e , f Reciprocal co-IP analysis of PRMT1 and FBXO7 in PRMT1 KD cells. The immunoblotting experiments were repeated three times with similar results. g Recombinant GST-FBXO7 protein was incubated with recombinant His-MBP-PRMT1 protein, followed by GST pulldown and immunoblotting analysis with GST and His antibodies. The immunoblotting experiments were repeated three times with similar results. h Schematic representation of full-length (FL) PRMT1 and different truncation mutants. i , j GFP-PRMT1 FL or indicated truncation mutants were co-expressed with FLAG-FBXO7 in HEK293T cells. FLAG-FBXO7 was immunoprecipitated with FLAG beads, followed by immunoblotting analysis of GFP-PRMT1 using GFP antibody. The immunoblotting experiments were repeated three times with similar results. k Schematic representation of full-length (FL) FBXO7 and different truncation mutants. UBL ubiquitin-like domain, FP FBXO7-PI31 dimerization domain, PRR proline-rich region. l , m FLAG-FBXO7 FL or indicated truncation mutants were co-expressed with GFP-PRMT1 in HEK293T cells. FLAG-FBXO7 was immunoprecipitated with FLAG beads, followed by immunoblotting analysis of GFP-PRMT1 using GFP antibody. The immunoblotting experiments were repeated three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Co-Immunoprecipitation Assay, Negative Control, Western Blot, Recombinant, Incubation, Immunoprecipitation

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a Immunoblotting analysis of PRMT1 (anti-PRMT1 antibody: Abcam, ab190892) and FBXO7 in FBXO7 KD cells treated with or without MG132 (25 μM, 6 h). The immunoblotting experiments were repeated three times with similar results. b Immunoblotting analysis of PRMT1 and GFP-FBXO7 in FBXO7-overexpressing cells treated with or without MG132 (25 μM) for 6 h. The immunoblotting experiments were repeated three times with similar results. c Immunoblotting analysis of PRMT1 and FBXO7 in FBXO7 KD cells treated with or without cycloheximide (CHX, 50 μg/mL) for the indicated time. Quantitation of PRMT1 protein level based on band intensity was shown (bottom). Data are presented as the mean ± SD ( n = 3 independent experiments with similar results). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. d Schematic representation of full-length (FL) FBXO7 and enzymatically dead mutant (deletion of F-box domain, shown as ΔF-box). e GFP-PRMT1 and HA-ubiquitin (HA-Ub) were co-expressed with FLAG-FBXO7 WT or enzymatically dead ΔF-box mutant in Huh7 cells. After MG132 (25 μM, 6 h) treatment, IP was performed with GFP antibody, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. f GFP-PRMT1 was co-expressed with HA-Ub in FBXO7 KD cells. After MG132 (25 μM, 6 h) treatment, IP was performed with GFP antibody, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. g FBXO7 KD cells were transfected with HA-Ub plasmid and treated with MG132 (25 μM, 6 h). IP was performed with IgG or PRMT1 antibody, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Western Blot, Quantitation Assay, Mutagenesis, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a Schematic of the workflow for identifying the ubiquitination sites of PRMT1 by mass spectrometry analysis. b Four potential lysine ubiquitination sites (K37, K82, K202, K325) in PRMT1 identified by mass spectrometry analysis. c GFP-PRMT1 (WT or indicated KR mutants) was co-expressed with HA-Ub in FBXO7 KD Huh7 cells rescued with or without FLAG-FBXO7. After MG132 (25 μM, 6 h) treatment, IP was performed with GFP antibody, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. d FLAG-FBXO7 was co-expressed with GFP-PRMT1 WT or K37R in Huh7 and PLC/PRF/5 cells, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. e FBXO7 KD cells were transfected with GFP-PRMT1 WT or K37R plasmid, and treated with or without cycloheximide (CHX, 50 μg/mL) for the indicated time. Immunoblotting analysis of GFP-PRMT1 and FBXO7 was performed. Quantitation of GFP-PRMT1 protein level based on band intensity was shown (bottom). Data are presented as the mean ± SD ( n = 3 independent experiments with similar results). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. f Mass spectrometry identification of K37 ubiquitination of PRMT1. All immunoblotting experiments were repeated three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Mass Spectrometry, Western Blot, Transfection, Plasmid Preparation, Quantitation Assay

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a , b PHGDH was immunoprecipitated in FBXO7 KD ( a ) or GFP-FBXO7-overexpressing ( b ) cells, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. c PHGDH was immunoprecipitated in FBXO7 and/or PRMT1 KD cells, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. d PHGDH was immunoprecipitated in cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. e Endogenous PHGDH was immunoprecipitated in HEK293T, Huh7, and PLC/PRF/5 cells with FBXO7 KD, followed by measurement of PHGDH activity. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. f GFP-FBXO7 plasmid was transfected in HEK293T cells stably expressing FLAG-PHGDH, followed by IP with FLAG beads and elution with FLAG peptides. PHGDH activity was then measured. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. g Endogenous PHGDH was immunoprecipitated in FBXO7 and/or PRMT1 KD cells, followed by measurement of PHGDH activity. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. h Endogenous PHGDH was immunoprecipitated in cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R, followed by measurement of PHGDH activity. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Immunoprecipitation, Western Blot, Activity Assay, Two Tailed Test, Plasmid Preparation, Transfection, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a Schematic of U-[ 13 C]-glucose incorporation into serine and glycine in cells. b, c Incorporation of U-[ 13 C]-glucose carbon into serine ( b ) and glycine ( c ) detected by LC–MS/MS in cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. d, e GSH ( d ) and ROS ( e ) levels in FBXO7 and/or PRMT1 KD cells cultured in CM or -SG medium. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. f ROS levels in cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R cultured in CM or -SG medium. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. g ROS levels in FLAG-FBXO7-overexpressing cells cultured in -SG medium in the presence or absence of NAC (5 mM). Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Cell Culture

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a Growth rates of FBXO7 and/or PRMT1 KD cells grown in CM or −SG medium. Data are presented as the mean ± SD ( n = 5 independent experiments). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. b Growth rates of cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R grown in CM or −SG medium. Data are presented as the mean ± SD ( n = 5 independent experiments). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. c Growth rates of FLAG-FBXO7-overexpressing cells grown in -SG medium in the presence or absence of NAC (5 mM). Data are presented as the mean ± SD ( n = 5 independent experiments). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. d Immunoblotting analysis of cleaved caspase 3 and caspase 3 in FLAG-FBXO7-overexpressing cells grown in −SG medium in the presence or absence of NAC (5 mM). The immunoblotting experiments were repeated three times with similar results. e , f FBXO7 and/or PRMT1 KD Huh7 cells were subcutaneously inoculated into nude mice fed with a +SG or −SG diet. Tumor image ( e ) and weight ( f ) were shown. Data are presented as the mean ± SD ( n = 6 mice). Statistical analysis in f was performed using the two-tailed Student’s t -test. g , h Huh7 cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R were subcutaneously inoculated into nude mice fed with a +SG or −SG diet. Tumor image ( g ) and weight ( h ) were shown. Data are presented as the mean ± SD ( n = 5 mice). Statistical analysis in h was performed using the two-tailed Student’s t -test. i Representative images of IHC staining for Ki-67, 8-oxo-dG, and cleaved caspase 3 in tumor xenografts in ( g ). Scale bars, 50 μm. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Western Blot, Two Tailed Test, Immunohistochemistry

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a , b Representative images ( a ) and quantitative analysis ( b ) of IHC staining for FBXO7 in HCC tissues and paired normal tissues ( n = 45 samples). Scale bars, 50 μm. Statistical analysis was performed using the paired two-tailed Student’s t -test. c , d Representative images ( c ) and quantitative analysis ( d ) of IHC staining for PRMT1 in HCC tissues and paired normal tissues ( n = 45 samples). Scale bars, 50 μm. Statistical analysis was performed using the paired two-tailed Student’s t -test. e and f Representative images ( e ) and quantitative analysis ( f ) of IHC staining for mePHGDH (R236me1) in HCC tissues and paired normal tissues ( n = 45 samples). Scale bars, 50 μm. Statistical analysis was performed using the paired two-tailed Student’s t -test. g Representative images of IHC staining with FBXO7, PRMT1, and mePHGDH (R236me1) antibodies in HCC tissues ( n = 45 samples). Scale bars, 50 μm. h Two-tailed Pearson correlation test analyzing the relationship between the IHC staining intensity of FBXO7 and PRMT1 in HCC tissues ( n = 45 samples). i Two-tailed Pearson correlation test analyzing the relationship between the IHC staining intensity of FBXO7 and mePHGDH (R236me1) in HCC tissues ( n = 45 samples). j Two-tailed Pearson correlation test analyzing the relationship between the IHC staining intensity of PRMT1 and mePHGDH (R236me1) in HCC tissues ( n = 45 samples). k IP and immunoblotting analysis with indicated antibodies in 6 HCC tissues (T1–T6). The immunoblotting experiments were repeated three times with similar results. l Two-tailed Pearson correlation test analyzing the relationship between the levels of FBXO7–PRMT1 interaction and PRMT ubiquitination in HCC tissues based on band intensity in k ( n = 6 samples). m Schematic model for the mechanism of FBXO7–PRMT1–PHGDH axis in regulating serine synthesis, oxidative stress, and HCC growth. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Immunohistochemistry, Two Tailed Test, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: PRMT1 binds and methylates TRAF6. A, Huh7.5 cells were transiently transfected with expression plasmids for PRMT1-GFP and TRAF6-FLAG where indicated. TRAF6-FLAG was immunoprecipitated (IP) using anti-FLAG affinity resin. Immunoprecipitated proteins were blotted (IB) for TRAF6 (anti-FLAG antibody) and PRMT1. B, Western blot analysis of endogenous PRMT1 and TRAF6 co-immunoprecipitated from Huh7.5 cells untreated or treated with FSL-1 (TLR6/2 ligand) for the indicated times using anti-TRAF6 antibody or IgG as a negative control. C, Huh7.5 cells were lysed, and arginine-methylated proteins were immunoprecipitated and analyzed by Western blot for the presence of methylated TRAF6, IRAK1, and SAM68 (positive control) using specific antibodies (L, load; IgG, nonspecific pulldown with mouse IgG; MeR, proteins immunoprecipitated with anti-methyl-arginine antibody). D, TRAF6 was immunoprecipitated and immunoblotted with methyl-arginine antibody from control cells or cells expressing wild type PRMT1 (P1, left) or cells expressing negative control (NC) or PRMT1-specific siRNA (right). Lower panels show the densitometry quantification of MeR/TRAF6 signal ratio from three independent immunoprecipitation experiments. **, p < 0.01. E, TRAF6 immunoprecipitation and methyl-arginine immunoblot of lysates from primary mouse hepatocytes expressing negative control (NC) or PRMT1 specific siRNA. F, Western blot analysis of endogenous PRMT1 and TRAF6 co-immunoprecipitated from THP-1 cells using anti-TRAF6 antibody or IgG as a negative control (left). Shown are Representative images of proximity ligation assays in THP-1 cells (right). TRAF6 interaction with PRMT1 was detected using anti-PRMT1 and anti-TRAF6 antibodies. Negative control, signal in the absence of primary antibodies. G, representative images of proximity ligation assays in THP-1 cells untreated or treated with AMI-1 for 16 h. TRAF6 methylation was detected using the combination of anti-TRAF6 and anti-methyl-arginine antibodies.

Article Snippet: In Vitro Activity Assays In vitro methylation assay was performed in a reaction mixture containing purified FLAG-TRAF6 (wt or mutants), 50 μ m SAM, 0.5 μg of recombinant human PRMT1 (Origene) in 1× PBS buffer.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Negative Control, Methylation, Positive Control, Ligation

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: PRMT1 activity is required for suppression of the TRAF6-dependent TLR pathway. A, Huh7.5 cells were transfected with HA-ubiquitin-Lys-63 (all lysines were substituted with arginine except Lys-63). Cells were otherwise untreated, pretreated with 10 μm AMI-1, or treated with negative control (NC) or PRMT1-specific siRNA. Lys-63-linked polyubiquitinated proteins were immunoprecipitated (IP) with anti-HA antibody, and the presence of TRAF6 was analyzed by Western blotting (IB). B, nuclear fractions were prepared from Huh7.5 or THP-1 cells treated as indicated with siRNA, the methylation inhibitor AMI-1 (10 μm), or the TLR6/2 ligand FSL-1. Immunoblots for NF-κB p65 are shown. C, NF-κB luciferase reporter assays of Huh7.5 cells expressing negative control (NC) or PRMT1-specific siRNA, expressing either wild type or dominant-negative PRMT1, or cells expressing TRAF6 dominant-negative construct treated with or without 10 μm AMI-1 as indicated. Transfection efficiency was controlled by expression of Renilla luciferase, and data are presented as the mean ± S.D. of relative luminescence. *, p < 0.05; **, p < 0.01. n ≥ 4. D, NF-κB luciferase reporter assays from RAW264.7 cells (control or pretreated with 10 μm AMI-1) or THP-1 cells expressing negative control (NC) or PRMT1-specific siRNA. Data are presented as the mean ± S.D. *p < 0.05. n ≥ 3. E, relative mRNA of NF-κB target genes TNFα, IL6, IRAK3 (IRAK-M), and TNFAIP3 (A20) in Huh7.5 cells, primary mouse hepatocytes, or primary mouse macrophages treated with or without AMI-1 for 16 h. Data are presented as the mean ± S.D. **, p < 0.01; *, p < 0.05. n = 3.

Article Snippet: In Vitro Activity Assays In vitro methylation assay was performed in a reaction mixture containing purified FLAG-TRAF6 (wt or mutants), 50 μ m SAM, 0.5 μg of recombinant human PRMT1 (Origene) in 1× PBS buffer.

Techniques: Activity Assay, Transfection, Negative Control, Immunoprecipitation, Western Blot, Methylation, Luciferase, Expressing, Dominant Negative Mutation, Construct

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: Identification of functional TRAF6 methylation sites. A, upper panel, in vitro methylation assay. Wild type or mutant TRAF6 purified from Huh7.5 cells was methylated in vitro by recombinant PRMT1 in the presence of 50 μm SAM and analyzed using anti-TRAF6 and anti-methyl-arginine antibody. Lower panel, in vitro ubiquitination assay. Untreated wild type or mutant TRAF6 or each TRAF6 methylated in vitro by PRMT1 as above was incubated in vitro with recombinant E1, E2, and NF-κ-B essential modulator (NEMO) added as a ubiquitination substrate. Polyubiquitinated NEMO was detected using specific antibody against polyubiquitin chains. MeR, proteins immunoprecipitated with anti-methyl-arginine antibody. B, NF-κB luciferase reporter assays from control cells or cells expressing wild type or mutant TRAF6 co-transfected with or without wild type PRMT1 where indicated. Data are presented as the mean ± S.D. *, p < 0.05, n = 3. C, NF-κB luciferase reporter assays. Control Huh7.5 cells or cells overexpressing JMJD6 were co-transfected with wild type or mutant constructs of TRAF6. *, methylation at this site was confirmed by mass spectrometry. Data are presented as the mean ± S.D. †, p < 0.05; ††, p < 0.01, n ≥ 3. D, relative levels of TNF-α mRNA in Huh7.5 cells transfected with indicated TRAF6 mutants with or without co-expression of JMJD6 as indicated. **, p < 0.01 compared with control; †, p < 0.05 compared with wild type TRAF6 alone. n = 3.

Article Snippet: In Vitro Activity Assays In vitro methylation assay was performed in a reaction mixture containing purified FLAG-TRAF6 (wt or mutants), 50 μ m SAM, 0.5 μg of recombinant human PRMT1 (Origene) in 1× PBS buffer.

Techniques: Functional Assay, Methylation, In Vitro, Mutagenesis, Purification, Recombinant, Ubiquitin Assay, Incubation, Immunoprecipitation, Luciferase, Expressing, Transfection, Construct, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: TRAF6 is demethylated after activation of TLR pathway. A, Western blot analysis of TRAF6 immunoprecipitated (IP) and immunoblotted (IB) with anti-TRAF6 and anti-methyl-arginine antibody (top left) or JMJD6 and PRMT1 protein levels (lower left) in Huh7.5 cells treated with FSL-1 for the indicated times. The right panel shows densitometry quantification of MeR/TRAF6 signal ratio and PRMT1 relative protein levels for three independent experiments. Data are presented as mean ± S.D. **, p < 0.01 (TRAF6 methylation); ††, p < 0.01 (PRMT1) compared with untreated cells. B, relative PRMT1 protein levels and relative TRAF6 methylation in THP-1 cells treated with LPS for the indicated times, measured by ELISA as described under “Experimental Procedures.” Data are presented as the mean ± S.D. **, p < 0.01 (TRAF6 methylation); †, p < 0.05; ††, p < 0.01 (PRMT1) compared with untreated cells. n = 3. C, control cells or cells overexpressing wild type PRMT1 were treated with or without FSL-1 for 30 min, and lysates were immunoprecipitated and immunoblotted with methyl-arginine antibody. Densitometry quantification of MeR/TRAF6 signal ratio is shown for three independent experiments. Data are presented as the mean ± S.D. *, p < 0.05; **, p < 0.01. n ≥ 3. MeR, proteins immunoprecipitated with anti-methyl-arginine antibody. D, cells were transfected with either negative control or JMJD6-specific siRNA and analyzed as in C. Densitometry quantification of MeR/TRAF6 signal ratio is shown for three independent experiments. Data are presented as the mean ± S.D. *, p < 0.05; **, p < 0.01. n ≥ 3. E, top two panels, TRAF6 was immunoprecipitated and immunoblotted with methyl-arginine antibody from primary mouse hepatocytes transfected with negative control (NC) siRNA or JMJD6 (J6)-specific siRNA and treated with FSL-1 ligand for indicated times. Bottom panel, Western blot of PRMT1 protein levels in cells treated with FSL-1 for the indicated times.

Article Snippet: In Vitro Activity Assays In vitro methylation assay was performed in a reaction mixture containing purified FLAG-TRAF6 (wt or mutants), 50 μ m SAM, 0.5 μg of recombinant human PRMT1 (Origene) in 1× PBS buffer.

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Methylation, Enzyme-linked Immunosorbent Assay, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: Impact of TRAF6 arginine methylation on the ligand-induced TLR response. A, THP-1-Lucia NF-κB reporter monocytes were transfected with negative control siRNA or JMJD6-specific siRNA and treated with ligands for different TLR molecules as indicated. Results of luciferase reporter assays are presented as the mean ± S.D. *, p < 0.05; **, p < 0.01 compared with negative control (NC) siRNA. n ≥ 3. B, luciferase reporter assays in Huh7.5 cells (left panel) or THP-1-Lucia NF-κB reporter monocytes (right panel) transfected with negative control siRNA or PRMT1-specific siRNA. Cells were treated with TLR ligands as indicated. Data are presented as the mean ± S.D. †, p < 0.05; ††, p < 0.01 compared with negative control siRNA. n ≥ 3. C, -fold increase in mRNA levels induced by FSL-1 exposure was determined in Huh7.5 cells treated with or without PRMT1 inhibitor AMI-1 for 16 h where indicated. *, p < 0.05; **, p < 0.01 compared with control; †, p < 0.05 compared with FSL-1 only. ICAM, intercellular adhesion molecule 1. D, FSL-1-induced -fold mRNA changes are shown in primary peritoneal macrophages. Cells were transfected with negative control (NC) siRNA and PRMT1-specific or JMJD6-specific siRNA where indicated. *, p < 0.05, compared with control; †, p < 0.05 compared with NC siRNA, n = 3. E, NF-κB luciferase reporter assays in Huh7.5 cells in the presence or absence of TLR6/2 ligand (1 ng/ml of FSL-1) that were transfected with negative control siRNA, PRMT1-specific siRNA, or both PRMT1- and JMJD6-specific siRNA. Data are presented as the mean ± S.D. *, p < 0.05; **, p < 0.01 compared with NC siRNA; ††, p < 0.01 compared with P1 siRNA. n = 3.

Article Snippet: In Vitro Activity Assays In vitro methylation assay was performed in a reaction mixture containing purified FLAG-TRAF6 (wt or mutants), 50 μ m SAM, 0.5 μg of recombinant human PRMT1 (Origene) in 1× PBS buffer.

Techniques: Methylation, Transfection, Negative Control, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: TRAF6 methylation correlates with TLR responses in humans. A, cytosolic and nuclear extracts were prepared from normal human liver tissue (liver transplant donor biopsy samples). Western blots were performed for PRMT1 and JMJD6 in cytosolic fractions and for NF-κB p65 in nuclear fractions. Data points represent values for individual liver specimens. n = 20. B, human peripheral blood monocytes were differentiated into macrophages and treated with or without AMI-1 for 16 h. Left panel, relative mRNA of TNFα in individual human macrophage preparations before (C) and after exposure to AMI-1. Right panel, -fold increase in TNFα mRNA 1 h after LPS exposure. Cells were pretreated with AMI-1 as indicated. Individual patient samples are connected by lines, n = 10. C, TRAF6 methylation was measured by ELISA as described under “Experimental Procedures.” Western blots were performed for JMJD6 protein concentration in cytosolic fractions from individual HBDM preparations. PRMT1 was measured using ELISA. n = 26. D, basal mRNA levels of TNFα and IL6 are plotted against relative TRAF6 methylation in individual HBDM preparations. E, -fold increase in IL6 mRNA levels is plotted against relative TLR4 expression or relative TRAF6 methylation in individual HBDM preparations. n = 26. Dependence of LPS-induced mRNA increases on TLR4 expression and TRAF6 methylation (6Tme) were statistically significant by multivariate linear regression, p = 0.0005 and 0.0017, respectively.

Article Snippet: In Vitro Activity Assays In vitro methylation assay was performed in a reaction mixture containing purified FLAG-TRAF6 (wt or mutants), 50 μ m SAM, 0.5 μg of recombinant human PRMT1 (Origene) in 1× PBS buffer.

Techniques: Methylation, Western Blot, Enzyme-linked Immunosorbent Assay, Protein Concentration, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: IRAK3 is responsible for ligand desensitization under low methylation conditions. A, Western blot analysis of IRAK-3 levels in Huh7.5 cells treated with 10 μm AMI-1 for 16 h with or without 1 ng/ml FSL-1 for the indicated times. B, analysis of human blood derived macrophages. Basal mRNA level of IRAK3 is plotted against PRMT1/JMJD6 (left) or relative TRAF6 methylation (right). -Fold increase in IL6 mRNA after 1 h of LPS treatment is plotted against IRAK3 protein concentration (bottom). Each point represents an individual HBDM preparation, n = 26. C, NF-κB luciferase reporter assays in THP-1 cells that were either untreated (control) or treated with TLR6/2 (FSL-1), TLR4 (LPS) or TLR1/2 (Pam3) ligands as indicated. Cells were first transfected with negative control (NC) siRNA, PRMT1-specific siRNA, IRAK3-specific siRNA, or both IRAK3 and PRMT1 siRNA. Data are presented as the mean ± S.D. **, p < 0.01. n ≥ 3. ns, not significant.

Article Snippet: In Vitro Activity Assays In vitro methylation assay was performed in a reaction mixture containing purified FLAG-TRAF6 (wt or mutants), 50 μ m SAM, 0.5 μg of recombinant human PRMT1 (Origene) in 1× PBS buffer.

Techniques: Methylation, Western Blot, Derivative Assay, Protein Concentration, Luciferase, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: A model of TRAF6 regulation by arginine methylation. In the basal state TRAF6 is associated with PRMT1, methylated, and inactive (State 1). TLR ligands activate TRAF6 and induce PRMT1 degradation. Reduction of PRMT1 levels allows JMJD6 to demethylate TRAF6 and results in the transition to a fully active form (State 2) leading to maximal NF-κB activation. The NF-κB-driven accumulation of inhibitory molecules, such as IRAK-3, eventually produces feedback inhibition reducing pathway activation. This allows the re-accumulation of PRMT1. Restoration of the PRMT1/JMJD6 ratio results in the re-methylation of TRAF6 and the return to the basal, inactive state.

Article Snippet: In Vitro Activity Assays In vitro methylation assay was performed in a reaction mixture containing purified FLAG-TRAF6 (wt or mutants), 50 μ m SAM, 0.5 μg of recombinant human PRMT1 (Origene) in 1× PBS buffer.

Techniques: Methylation, Activation Assay, Inhibition

Journal: Oncogenesis

Article Title: PRMT1 promotes neuroblastoma cell survival through ATF5

doi: 10.1038/s41389-020-0237-9

Figure Lengend Snippet: a , b Gene ontology analyses of differentially expressed genes in SK-N-BE(2)C cells after PRMT1 depletion. c qRT-PCR (top) and Western blot (bottom) of SK-N-BE(2)C shPRMT1-D6 with or without Dox. Data are mean ± SD ( n = 3) relative to without Dox. d Western blot of SK-N-BE(2)C shPRMT1-D6 cells transduced with pOZ vector or pOZ-ATF5. e Cell proliferation assay of SK-N-BE(2)C shPRMT1-D6 cells transduced with pOZ vector or pOZ-ATF5 with or without Dox. Data are mean ± SD ( n = 3) relative to day 1. f Caspase-3/7 activity assay of SK-N-BE(2)C shPRMT1-D6 cells transduced with pOZ vector or pOZ-ATF5 with or without Dox. Data are mean ± SD ( n = 3) relative to pOZ without Dox. g qRT-PCR of cells transfected with ON-TARGETplus human siPRMT1 pool or ON-TARGETplus Non-targeting pool. HPRT1 was used as an internal control. Data are mean ± SD ( n = 3) relative to cells transfected with Non-targeting pool. h UCSC genome browser tracks of the ATF5 gene locus showing PRMT1 binding with two different antibodies. i ChIP analysis of PRMT1 at ATF5 and CITED2 gene promoters in SK-N-BE(2)C cells. j ChIP analysis of H4R3me2a at ATF5 and CITED2 gene promoters in SK-N-BE(2)C shPRMT1-D6 cells with or without Dox. Data are mean ± SD ( n = 3). ** P < 0.01.

Article Snippet: ChIP assays were performed as described previously using antibodies specific for PRMT1 or H4R3me2a (Active Motif).

Techniques: Quantitative RT-PCR, Western Blot, Transduction, Plasmid Preparation, Proliferation Assay, Activity Assay, Transfection, Control, Binding Assay

Journal: Oncotarget

Article Title: Tumor suppressor BTG1 promotes PRMT1-mediated ATF4 function in response to cellular stress

doi: 10.18632/oncotarget.6519

Figure Lengend Snippet: ( A ) BTG1 binds to ATF4. HEK293 cells were transfected with expression plasmids encoding HA-ATF4 and FLAG-BTG1 and treated for 24 hrs with 5 μM of the proteasome inhibitor MG132. Protein lysates were generated and subjected to immunoprecipitation (IP) with FLAG antibody (Ab). Immunoblot demonstrates expression of BTG1 using a FLAG-Ab. ( B ) PRMT1 binds to ATF4 in a BTG1-dependent manner. WT and Btg1 −/− MEFs were treated with glutamine starvation for 16 h. Protein lysates were generated and subjected to IP with PRMT1 Ab. Immunoblot demonstrates expression of ATF4. ( C ) Mapping of arginine residues found in ATF4. ( D ) ATF4 is methylated by PRMT1 at amino acid (aa) residue 239. GST-purified ATF4 WT and various ATF4 deletion mutants (top panel) and arginine mutants (bottom panel) were subjected to in vitro methylation assays together with purified PRMT1 and S-adenosyl methionine as a methyl donor. Proteins were resolved by SDS-PAGE, stained with Coomassie blue (ATF4 input), dried and analyzed by autofluorography. Mutation of arginine 239 abolishes methylation of ATF4.

Article Snippet: As much as 1 μg of purified recombinant PRMT1 (ProSpec Bioscience) was incubated with 5 μg of purified GST-fusion ATF4 in the presence of radioactive methyl donor [3H-methyl] -S-Adenosyl Methionine (55–85Ci (2.03–3.15TBq)/mM; GE Healthcare Life Science) in a total of 30 μl of PBS for 2 h at 30°C.

Techniques: Transfection, Expressing, Generated, Immunoprecipitation, Western Blot, Methylation, Purification, In Vitro, SDS Page, Staining, Mutagenesis

Journal: Oncotarget

Article Title: Tumor suppressor BTG1 promotes PRMT1-mediated ATF4 function in response to cellular stress

doi: 10.18632/oncotarget.6519

Figure Lengend Snippet: ( A ) In response to sustained stress conditions, BTG1 recruits PRMT1 to methylate ATF4, - indicated by ‘Me’- which promotes transcription of a subset of ATF4 target genes, leading to increased apoptosis. ( B ) In the absence of BTG1, PRMT1 no longer binds to and methylates ATF4, shifting the balance from pro-apoptotic to pro-survival. As a consequence, loss of BTG1 function promotes (tumor) cell survival.

Article Snippet: As much as 1 μg of purified recombinant PRMT1 (ProSpec Bioscience) was incubated with 5 μg of purified GST-fusion ATF4 in the presence of radioactive methyl donor [3H-methyl] -S-Adenosyl Methionine (55–85Ci (2.03–3.15TBq)/mM; GE Healthcare Life Science) in a total of 30 μl of PBS for 2 h at 30°C.

Techniques:

Journal: Molecular cell

Article Title: Mechanistic view of hnRNPA2 low complexity domain structure, interactions, and phase separation altered by disease mutation and arginine methylation

doi: 10.1016/j.molcel.2017.12.022

Figure Lengend Snippet: Key Resources Table

Article Snippet: Methylation by PRMT1 Low complexity domain methylated samples were generated by mixing purified 100 μM MBP-A2 LC with 100 μL of recombinant His-MBP tagged human PRMT1 (AtGen) and excess SAM and incubating overnight at 37°C.

Techniques: Recombinant, Software