human ube1  (Boston Biochem)

Journal: bioRxiv

Article Title: Parp7 generates an ADP-ribosyl degron that controls negative feedback of androgen signaling

doi: 10.1101/2024.12.21.629908

Figure Lengend Snippet: DTX2 is the E3 ligase for ADP-ribosylated AR. A, Immunoblot detection of Flag-AR and AR-ADPr (by FL-AF1521) in PC3-AR cells with siRNA knockdowns (total 4-day knockdown) of the selected relevant E3 ligases (DTX1, DTX2, DTX4, HUWE1, RNF146, SPOP, TRIP12 and UBR5), treated with R1881 for 21 hr before cell harvest. The AR/TUB and AR-ADPr/AR ratios for each lane are presented below the blot. B, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR cells with siDTX2 knockdown, treated with R1881 for times indicated on the panel. The AR-ADPr/AR ratio for each lane is presented below the blot. C, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR siCTRL and siDTX2 cell extracts and AF1521 bound fraction. Cell extracts from PC3-AR siCTRL and siDTX2 cells treated with R1881 for 6 hr were combined with AF1521 beads for the enrichment of ADP-ribosylated proteins. D, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR siCTRL/siDTX2 and PC3-AR HA- PARP7 cell extracts and GSH beads bound fraction. Cell extracts from PC3-AR siCTRL/siDTX2 and PC3-AR HA-PARP7 cells treated with R1881 for 6 hr were combined with GSH beads loaded with GST- DTX2-RD or GST-DTX2-RD mut for the enrichment of proteins recognized by DTX2 DTC domain. E, Diagrams of DTX2-RD and DTX2-RD mut . Three loss of function mutations in the DTC domain of DTX2-RD mut (S568A, H582A, and H594A) are indicated with red asterisk. F, Schematic diagram of AR protein preparation as a substrate for biochemical reactions. Cell extracts from PC3-AR siDTX2 cells treated (left) or untreated (right) with R1881 for 6 hr were combined with M2 beads for immunoprecipitation. The purified protein from the preparation with R1881 treatment was used for experiments in panels G and H, and the purified protein from the preparation without R1881 treatment was used only in panel H. G, Immunoblot detection of Flag-AR and AR-ADPr from the ubiquitylation assay on AR protein prepared with siDTX2 and R1881 treatment (panel F, left). The ubiquitylated products (Ub product, red bracket) are labeled for Flag-AR and AR-ADPr detection. All reactions contained AR-ADPr (R1881 treated samples), ATP, Ub, E1 and E2. For DTX2-RD status (dropout or DTX2-RD mut ), refer to labels. H, Immunoblot detection of Flag-AR, AR-ADPr, and GST-DTX2-RD from the ubiquitylation assay on AR protein prepared with siDTX2 transfection, and with or without R1881 treatment (panel F). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The dropouts of the ubiquitylation assay components (Ub, E1, E2, and 30°C incubation) are indicated on the labels. The T7-Ubiquitin (T7-Ub) was detected by Ponceau staining. Lane numbers are indicated below the blot. I, Bar plots showing the results of an RT-qPCR experiment in PC3-AR siCTRL/siDTX2 cells untreated (grey), treated with R1881 (purple), and cotreated with R1881 and RBN2397 (blue). The y-axis represents the relative expression normalized to the GUS housekeeping gene, and the x-axis represents the siRNA used. The p-values from the Welch’s t-test for comparisons between corresponding conditions in siCTRL and siDTX2 are indicated on the plots. Error bars represent standard deviation (n = 3; n represents number of biological replicates).

Article Snippet: Ubiquitylation assays were performed at 30°C for 30 min with 1 mM ATP, 100 g/ml Ub (T7-Ub or bovine Ub (Sigma U6253)), 5 g/ml each of UB E1 (R&D Systems E-304) and UB E2 (His-UbcH5C, R&D Systems E2-627), and 20 g/ml GST-DTX2 RD in the buffer E (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM DTT, 1 µg/ml each of A/L/P and 0.1 mg/ml BSA).

Techniques: Western Blot, Knockdown, Immunoprecipitation, Purification, Ubiquitin Assay, Labeling, Transfection, Incubation, Staining, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Parp7 generates an ADP-ribosyl degron that controls negative feedback of androgen signaling

doi: 10.1101/2024.12.21.629908

Figure Lengend Snippet: DTX2 conjugates ubiquitin to AR through ADP-ribose A, Immunoblot detection of Fluorescein (FITC) and T7-Ubiquitin (T7-Ub) from the ubiquitylation assay on FITC-AR(C284) or FITC-AR(C284 ADPr ) peptides. The labels indicate from the top: the substrate used (FITC-AR(C284) or FITC-AR(C284 ADPr ) peptides), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay (all reactions contained ATP, T7-Ub, E1 and E2, for DTX2-RD dropout, refer to labels), and the post- ubiquitylation assay treatments (NUDT16 or Mg 2+ buffer). Lane numbers are indicated below the blot. B, Schematic diagram representing FITC-AR(C284 ADPr ) peptide conjugated to ubiquitin (Ub). Indicated with red scissors are bonds within the ADP-ribose structure cleaved by NUDT16 and USP2. C, Schematic of the ubiquitylation assay workflow for panels D and E. D, Immunoblot detection of Flag-AR and AR-ADPr (by FL-AF1521) from the ubiquitylation assay on AR protein prepared with siDTX2 transfection and R1881 treatment (refer to panel 6f for sample preparation workflow). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The labels separated by black lines indicate sequential steps from top to bottom. From the top, the labels indicate the substrate used (AR-ADPr), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay, and the post-ubiquitylation assay treatments (USP2-CD, NUDT16, or Mg 2+ buffer). Lane numbers are indicated below the blot. E, Immunoblot detection of Flag-AR and AR-ADPr from the ubiquitylation assay on AR protein prepared with siDTX2 and R1881 treatment (refer to panel 6f for sample preparation workflow). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The labels separated by black lines indicate sequential steps from top to bottom. From the top, the labels indicate the substrate used (AR-ADPr), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay, and the post- ubiquitylation assay treatments (USP2-CD, NUDT16, or Mg 2+ buffer). Lane numbers are indicated below the blot. F, Scatter plots depicting the correlation between PARP7 (left) or DTX2 (right) mRNA expression and the response to androgen pathway activity calculated by PARADIGM in primary prostate cancer patients from TCGA-PRAD cohort. Each dot represents one patient (n = 478, n represents number of patients). Pearson correlation coefficients and corresponding p-values are indicated on the plots. G, Kaplan-Meier plot depicting progression-free interval (PFI) in primary prostate cancer patients from the TCGA-PRAD cohort, stratified by PARP7 expression levels. The red line represents patients with high PARP7 expression (top 25%), and the green line represents patients with low PARP7 expression (bottom 25%). The X-axis represents time (days), and the Y-axis represents the progression-free interval probability. The interval distributions were compared using the log-rank test, with the p-value indicating statistical significance. Dotted lines represent the 95% confidence interval.

Article Snippet: Ubiquitylation assays were performed at 30°C for 30 min with 1 mM ATP, 100 g/ml Ub (T7-Ub or bovine Ub (Sigma U6253)), 5 g/ml each of UB E1 (R&D Systems E-304) and UB E2 (His-UbcH5C, R&D Systems E2-627), and 20 g/ml GST-DTX2 RD in the buffer E (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM DTT, 1 µg/ml each of A/L/P and 0.1 mg/ml BSA).

Techniques: Western Blot, Ubiquitin Assay, Transfection, Sample Prep, Labeling, Expressing, Activity Assay

Journal: Nucleic Acids Research

Article Title: PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling

doi: 10.1093/nar/gkae918

Figure Lengend Snippet: PCNA ubiquitylation facilitates RNF168-binding. ( A ) ITC was conducted by titrating synthetic peptide (p21, GRKRRQTSMTDFYHSKRRLIFS-amide where underlined residues denote PIP box; syringe, 150–300 μM) into a solution of PCNA (cell; 30 μM) in TBS. Control experiments using peptide injected into buffer alone showed minimal heats with no evidence of titration. Analysis of the isotherm yielded K d = 76.3 nM and stoichiometry = 0.93 (Microcal Origin software). ( B ) Replicate plates of H1299 cells were infected with adenovirus vectors encoding FLAG-RNF168 WT and HA-PCNA (or an ‘empty’ adenovirus vector for control). Thirty-six hours post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-FLAG antibody in the presence of different concentrations of peptides corresponding to the p21 or Polη PIP boxes. Anti-FLAG immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies. ( C ) Replicate plates of H1299 cells were co-infected with adenovirus vectors encoding HA-PCNA and FLAG-RNF168 WT or HA-PCNA and RNF168 ΔDPIP. Some cells received an ‘empty’ adenovirus vector instead of HA-PCNA (for control). Thirty-six hours post-infection, some plates were treated with 2 mM HU for 2 h. Chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody in the presence or absence of the p21 PIP box peptide (1 mM). Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies. ( D ) Replicate plates of H1299 cells were infected with adenovirus vectors encoding wild-type PCNA (HA-PCNA WT), ubiquitylation-resistant PCNA (HA-PCNA K164R) or a PCNA-ubiquitin fusion (HA-PCNA-Ub) in combination with FLAG-RNF168 WT adenovirus. Some cells received an ‘empty’ adenovirus vector instead of HA-PCNA viruses(for control). Thirty-six hours post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody. Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies. ( E ) Replicate plates of H1299 cells were transfected with expression plasmids encoding WT or mutant forms of RNF168. The transfected cultures were then infected with adenovirus vector encoding wild-type PCNA (HA-PCNA WT) or with an ‘empty’ adenovirus vector (for control). Thirty-six hours post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody. Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies.

Article Snippet: Ubiquitylation assays were performed in 25 μl reactions in which the components were added in the following order: dd H2O, 1× Energy regeneration solution (#B-10 R&D systems), 75 mM ubiquitin (#U-100H R&D systems), 1.6 mM FLAG-PCNA substrate (expressed and purified in bacteria), 0.1 mM E1 Ubiquitin Activating Enzyme (#E-304 R&D systems), 0.2 mM E2 conjugase (UbcH5c, #E2-627 R&D systems or RAD6 #E2-613 R&D Systems), 0.2 mM E3 ligase (recombinant bacterial RAD18-RAD6 complex or RNF168 both purified in-house).

Techniques: Binding Assay, Control, Injection, Titration, Software, Infection, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot, Transfection, Expressing, Mutagenesis