Fig 1: TRIP12 Interacts with and Poly-Ubiquitylates PARP1 in a PAR- and WWE-Dependent Manner(A) HEK293T cells were transfected with the indicated plasmids for co-immunoprecipitation (coIP) experiments, with or without prior PARPi treatment (olaparib: 10 µM, 1 h). FLAG-PARP1 was immunoprecipitated and the interaction with GFP-PARP1 was analyzed by western blot. IP samples were adjusted based on input levels of PARP1 to correct for the effect of TRIP12 on PARP1 abundance.(B) In vitro interaction assay of purified GST, GST-TRIP12-WWE wild type (WT), and GST-TRIP12-WWE R869A mutant with PARP1 and auto-PARylated PARP1. Recombinant purified PARP1 was incubated or not with a low (20 µM, +) or high (200 µM, ++) concentration of NAD+ for 15 min at 30°C to induce PARP1 auto-PARylation prior to the GST interaction assay. PARP1 and PAR binding to the purified GST-fusion proteins was assessed by western blot.(C) Ex vivo interaction assay of purified GST, GST-TRIP12-WWE WT, and GST-TRIP12-WWE R869A mutant with whole cell lysates from HeLa cells, which were exposed to 1-mM H2O2 for 15 min prior to cell lysis to induce PAR formation. PARG was depleted from these cells by siRNA to avoid the PARG-mediated rapid degradation of PAR. PARP1 and PAR binding to the purified GST-fusion proteins was assessed by western blot.(D) In vitro ubiquitylation assay with purified E1 (UBE1), E2 (UBE2L3), E3 (FLAG-TRIP12 WT, WWE mutant (R869A), or HECT mutant (C2034A) purified from HEK293T cells and ubiquitin, using auto-PARylated PARP1 as a target protein. PARP1 ubiquitylation was assessed by western blot with anti-ubiquitin antibody. TRIP12 levels were assessed by running the supernatant from the in vitro reaction via western blot.(E) In vivo ubiquitylation assay in HEK293T cells expressing FLAG-PARP1 and GFP-TRIP12 WT, WWE mutant (R869A), or HECT mutant (C2034A) together with HA-ubiquitin. FLAG-PARP1 was immunoprecipitated and PARP1 ubiquitylation was assessed by western blot. IP samples were adjusted based on input levels of PARP1 to correct for the effect of TRIP12 on PARP1 abundance.(F) In vitro ubiquitylation assay to monitor TRIP12 activity in absence or presence of purified PAR chains. FLAG-TRIP12 WT or the PAR-binding-deficient WWE mutant (R869A) were purified from HEK293T cells and incubated with E1 (UBE1), E2 (UBE2L3), and ubiquitin with or without different amounts of purified PAR chains as indicated. Auto-ubiquitylation of TRIP12 was assessed by western blot.See also Figure S3.
Fig 2: TRIM28 mediates SIRT1 ubiquitination and proteasomal degradation. (A) HeLa cells were transfected with siTRIM28 for two consecutive days to inhibit TRIM28 expression. CHX chase experiments were performed in control HeLa cells and TRIM28 knockdown cells treated with DMSO or DOX (5µM) for 16 hours before addition of CHX (20nM) at indicated times. SIRT1 protein level was measured by western blot analysis. The blot is representative of three different independent experiments. (B) Quantification of SIRT1 protein degradation rate in (A). The quantification was done by the Fiji software. Data are presented as means ± SEM (n=3). (C) HEK293T cells were transfected with siTRIM28 previously for one day, followed by overexpression of FLAG-SIRT1 and HA-Ub for 24 h. The cells were harvested after MG132 (10µM) treatment for 2 h and lysed with denature IP lysis buffer, before immunoprecipitation with anti-FLAG beads. (D) Wild-type HeLa cells, TRIM28 KO HeLa cells and TRIM28 KO cells transfected with HA-TRIM28 or HA-TRIM28C65/68A were collected and subjected to western blot analysis with the relevant antibodies. (E) Same groups of cells in (D) were treated with DOX (5µM) and MG132 (10µM) for 8 h. The cells were lysed with denature IP lysis buffer, before immunoprecipitation with gfp-trap. (F) In vitro ubiquitination assay was performed using recombined His-SIRT1 protein and FLAG-TRIM28 pulled-down by FLAG-tagged agarose in HEK293T cells overexpressing FLAG-TRIM28. MYC-Ubiquitin, ATP, UBE1 and UBE2L6 (ubiquitination E2 conjugation enzyme) were used in the system and ubiquitination level was analyzed by western blot with the anti-His antibody. *, nonspecific bands. See also Figure S2.
Fig 3: Phospho-ubiquitinated human Parkin activates Parkin in trans. (A) Overview of experiments to detect in trans activation of Parkin. (B) Preparation of phospho-ubiquitinated Parkin. Recombinant HA-Parkin purified from bacteria was subjected to the ubiquitination assay in the presence of recombinant TcPINK1 WT or KD. Phospho-ubiquitination of Parkin was confirmed by western blot using anti-pUb. (C) MBP-Parkin ubiquitination assay using FLAG-ubiquitin, UBE1 and UbcH7 in the presence of HA-Parkin prepared as in (B). Input; MBP-Parkin without HA-Parkin. (D) Detection of Parkin self-activation by Ub-VS in the presence of phospho-ubiquitinated Parkin. MBP-Parkin was incubated with HA-Parkin [prepared as in (B)] for 10 min. MBP-Parkin~Ub; MBP-Parkin with Ub-VS covalently bound to its catalytic cysteine. Two replications were performed for each experiment in (C) and (D), and the representative results are shown.
Fig 4: AF enhances UBA1 interaction with most of the ubiquitin-conjugating E2s.a AF enhances UBA1 interactions with 20 E2s in cells. The heatmap was obtained by two independent ani-HA-UBA1 coIP followed by protein identification and quantification by mass spectrometry. The first four columns represent the summary p-value (columns 1,2) and fold change (columns 3,4) for control versus AF treatment groups. Columns 4–8 represent the UBA1-associated protein levels, calculated as an average from quadruplicate samples in each experimental run. b Guide tree of 36 human E2 ubiquitin-conjugating enzymes. Multiple sequence alignment and Guide tree of the 36 E2 proteins was performed by Clustal Omega39. The E2s in two clusters in red dash-line rectangles are E2s whose interactions with UBA1 were enhanced by AF as shown in (a). The three E2s underlined are exceptions. The number following the E2s is indicative of the evolutionary distance between the sequences. c Validation of AF-enhanced UBA1-E2 interactions in cells by anti-HA-UBA1 coIP as in (a) followed by IB. d Validation of AF-enhanced UBA1-E2 interactions in cells by anti-UBA1 coIP as in (a) followed by IB. e C1039A mutation diminishes UBA1 interactions with E2s. f AF does not promote the UBA6 interaction with UBE2Z in a GST-UBA6 pull-down assay. g AF does not affect the UBA2 interaction with UBE2I as revealed by anti-HA-UBE2I coIP. Source data are provided as a Source Data file.
Fig 5: AF facilitates ubiquitin charging to E2s.The effects of AF on ubiquitin charging in vitro to UBA1 (a), UBE2G2 (b, c), and UBE2D1 (d). The assays were performed using purified recombinant proteins. 250 nM UBA1 alone or in combination with 4 µM His-E2s (His-UBE2G2 or His-UBE2D1) were treated with increasing concentrations of AF in reaction buffer for 1 min. ATP (50 µM) was added to initiate the reaction. The reactions proceeded at 15 oC for 45 s and stopped by adding non-reducing loading buffer. 5 mM DTT was used to disrupt the thioester bond that links ubiquitin to the active cysteine of E2s. Source data are provided as a Source Data file.
Supplier Page from R&D Systems, a Bio-Techne Brand for Ubiquitin-activating Enzyme/UBE1 Protein
Available conjugates: Sizes Available: 25 ug