lysine less biotinylated ubiquitin  (R&D Systems)

Journal: The Journal of biological chemistry

Article Title: β-arrestin1 is an E3 ubiquitin ligase adaptor for substrate linear polyubiquitination.

doi: 10.1016/j.jbc.2023.105474

Figure Lengend Snippet: Figure 1. In vitro reconstitution of STAM1 ubiquitination by AIP4. A, STAM1 ubiquitination by AIP4 was reconstituted in vitro and performed in the presence of increasing concentrations of βarrestin1. Ubiquitination reactions comprised of E1(Ube1, 42 nM), E2 (UbcH7, 350 nM), E3 (AIP4, 48.5 nM), ubiquitin (11.6 μM), DTT (1 mM), ATP (1 mM) plus STAM1 (42 nM), and varying concentrations of βarr1 (0 nM, 20 nM, 40 nM, 80 nM, 160 nM, or 320 nM) in 40 μl. Reactions were incubated for 90 min at 37 C and terminated with 40 μl 2× sample buffer. Equal volumes were analyzed by 7% SDS-PAGE and immunoblotting with the indicated antibodies. Polyubiquitinated [Ub(n)] and unmodified STAM1 are indicated. Immunoblots are from one representative experiment. STAM1 ubiquitination was quantified from the STAM (B) and ubiquitin (C) immunoblots using densitometry and shown as line graphs. Data were expressed relative to the signal at 300 nM βarr1 and represent the mean ± S.D. from three independent experiments. Curves were fitted by nonlinear regression, Michaelis–Menten (GraphPad Prism). AIP4, atrophin-interacting protein 4; STAM, signal-transducing adaptor molecule.

Article Snippet: UbcH7 (E2; cat# E2-640–100), lysine-less ubiquitin (0K-Ub; cat# UM-NOK) and lysine-less biotinylated ubiquitin (0K-Ub biotin; cat# UB-560) were from R&D Systems.

Techniques: In Vitro, Ubiquitin Proteomics, Incubation, SDS Page, Western Blot

Journal: The Journal of biological chemistry

Article Title: β-arrestin1 is an E3 ubiquitin ligase adaptor for substrate linear polyubiquitination.

doi: 10.1016/j.jbc.2023.105474

Figure Lengend Snippet: Figure 2. Analysis of STAM1 ubiquitination with a STAM1 binding-deficient variant of β-arrestin1. A, ubiquitination reactions as described in Experimental procedures were incubated with 40 nM WT β-arr1 (WT) or STAM binding-deficient variant βarr1-4A (4A) in the presence or absence of 1 mM ATP. Reactions were immediately (0 min) terminated or after incubation at 37 C for 20 min in 2× sample buffer. Reactions were analyzed by 7% SDS-PAGE and immunoblotting with the indicated antibodies. Polyubiquitinated [Ub(n)] and unmodified STAM1 are indicated. Immunoblots are from one repre- sentative experiment. STAM1 ubiquitination was quantified from the STAM (B) and ubiquitin (C) immunoblots using densitometry and shown as bar graphs. Data were expressed relative to the signal with WT βarr1 incubated for 20 min and represent the mean ± S.D. from three independent experiments. D, binding analysis of AIP4 to WT or 4A βarr1. Equal amounts (500 ng) of purified WT or 4A βarr1 were incubated with equimolar amounts (1 μM) of GST-AIP4 and GST immobilized on glutathione-Sepharose resin. Bound βarr1 was detected by immunoblotting. A small fraction (1%) of purified protein is shown as input. Blots were stained with Ponceau-S to show the level of GST-AIP4 and GST used in the binding reactions. Blots are from one representative experiment. E, bound βarr1 was quantified using densitometry, normalized to GST, and shown as a bar graph. Data were expressed relative to the WT βarr1 signal and represent the mean ± S.D. from four independent experiments. Data were analyzed by an unpaired t test using GraphPad Prism. The adjusted p values are indicated. AIP4, atrophin-interacting protein 4; n.s., not significant; STAM, signal-transducing adaptor molecule.

Article Snippet: UbcH7 (E2; cat# E2-640–100), lysine-less ubiquitin (0K-Ub; cat# UM-NOK) and lysine-less biotinylated ubiquitin (0K-Ub biotin; cat# UB-560) were from R&D Systems.

Techniques: Ubiquitin Proteomics, Binding Assay, Variant Assay, Incubation, SDS Page, Western Blot, Staining

Journal: The Journal of biological chemistry

Article Title: β-arrestin1 is an E3 ubiquitin ligase adaptor for substrate linear polyubiquitination.

doi: 10.1016/j.jbc.2023.105474

Figure Lengend Snippet: Figure 3. Mass spectrometry analysis of STAM1 ubiquitination. A, workflow of mass spectrometry analysis of STAM1 ubiquitination. Ubiquitination reactions were as described in Experimental procedures (1), trypsin digested (2), analyzed by liquid chromatography tandem mass spectrometry (LC-MS) (3) and peptide identification (4). B, schematic of STAM1 and ubiquitin summary of mass spectrometry results. The green color corresponds to peptide coverage of STAM1 or ubiquitin that was observed in the mass spectrometry results, while the gray color corresponds to areas that were not covered. STAM has 29 lysine residues (indicated by ticks), with seven lysine residues identified to be modified by ubiquitin are indicated by magenta ticks. Lysine 136 (K136) is indicated and is located within the VHS (Vps27, HRS, STAM) domain. The UIM (ubiquitin interacting motif), SH3 (Src-homology domain 3) and the coiled-coil domains are shown. Ubiquitin has seven lysine residues (indicated by ticks); modified residues are indicated (K11, K27, K29, K48, and K63). The first methionine on ubiquitin was also modified by ubiquitin (M1). The figure was created with BioRender. STAM, signal-transducing adaptor molecule.

Article Snippet: UbcH7 (E2; cat# E2-640–100), lysine-less ubiquitin (0K-Ub; cat# UM-NOK) and lysine-less biotinylated ubiquitin (0K-Ub biotin; cat# UB-560) were from R&D Systems.

Techniques: Mass Spectrometry, Ubiquitin Proteomics, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of biological chemistry

Article Title: β-arrestin1 is an E3 ubiquitin ligase adaptor for substrate linear polyubiquitination.

doi: 10.1016/j.jbc.2023.105474

Figure Lengend Snippet: Figure 4. Evaluation of lysine 136 on STAM1 ubiquitination with WT or lysine-less ubiquitin. Ubiquitination reactions as described in Experimental procedures were incubated with WT STAM1 (WT) or variant in which lysine 136 has been substituted with an arginine (K136R) and with WT ubiquitin (WT Ub) (A) or lysine-less ubiquitin (0K Ub) (D). Reactions were performed in the presence of 40 nM βarr1 and with or without 1 mM ATP/MgCl2. Reactions were immediately (0 min) terminated in 2× sample buffer or after incubation at 37 C for 20 min (A) or 60 min (B). Reactions were analyzed by 7% SDS-PAGE and immunoblotting with the indicated antibodies. Polyubiquitinated [Ub(n)], unmodified and monoubiquitinated (mono-Ub) STAM1 are indicated. Immu- noblots are from one representative experiment. STAM1 ubiquitination was quantified from the STAM (B and E) and ubiquitin (C and F) immunoblots using densitometry and shown as bar graphs. Data were expressed relative to the signal with WT STAM1 and represent the mean ± S.D. from three independent experiments. G, purified βarr1 (0.1 μM) was incubated without (empty resin; n/a) or with immobilized STAM1-His (3 μM) WT or K136R variant for 20 min at 37 C. Complexes were analyzed by immunoblotting. Immunoblots are from a representative experiment. H, bound STAM1 normalized to βarr1 was quantified using densitometry and shown as bar graphs. Data represent the mean ± S.D. from three independent experiments and are expressed relative to the fraction of STAM1 bound to βarr1. Data were analyzed by an unpaired t test using GraphPad Prism. Adjusted p values are indicated. n.s., not significant. STAM, signal-transducing adaptor molecule.

Article Snippet: UbcH7 (E2; cat# E2-640–100), lysine-less ubiquitin (0K-Ub; cat# UM-NOK) and lysine-less biotinylated ubiquitin (0K-Ub biotin; cat# UB-560) were from R&D Systems.

Techniques: Ubiquitin Proteomics, Incubation, Variant Assay, SDS Page, Western Blot

Journal: The Journal of biological chemistry

Article Title: β-arrestin1 is an E3 ubiquitin ligase adaptor for substrate linear polyubiquitination.

doi: 10.1016/j.jbc.2023.105474

Figure Lengend Snippet: Figure 5. Analysis of STAM1 ubiquitination by preactivated β-arrestin1. Ubiquitination reactions as described in Experimental procedures were incubated with 40 nM WT βarr1 (WT) or preactivated variant of βarr1 (R169E) in the presence or absence of 1 mM ATP with WT ubiquitin (WT Ub) (A) or lysine-less ubiquitin (0K Ub) (D). Reactions were terminated in 2× sample buffer immediately (0 min) or after incubation at 37 C for 60 min. Reactions were analyzed by 7% SDS-PAGE and immunoblotting. Polyubiquitinated [Ub(n)], unmodified and monoubiquitinated (mono-Ub) STAM1 are indicated. Nonspecific bands are indicated. Data are representative of three independent experiments. STAM1 ubiquitination was quantified from the STAM (B and E) and ubiquitin (C and F) immunoblots using densitometry and shown as bar graphs. Data were expressed relative to the signal with WT βarr1 and represent the mean ± S.D. from three independent experiments. Data were analyzed by an unpaired t test using GraphPad Prism. Adjusted p values are indicated. STAM, signal-transducing adaptor molecule.

Article Snippet: UbcH7 (E2; cat# E2-640–100), lysine-less ubiquitin (0K-Ub; cat# UM-NOK) and lysine-less biotinylated ubiquitin (0K-Ub biotin; cat# UB-560) were from R&D Systems.

Techniques: Ubiquitin Proteomics, Incubation, Variant Assay, SDS Page, Western Blot

Journal: The Journal of biological chemistry

Article Title: β-arrestin1 is an E3 ubiquitin ligase adaptor for substrate linear polyubiquitination.

doi: 10.1016/j.jbc.2023.105474

Figure Lengend Snippet: Figure 7. Evaluation of lysine 136 on STAM1-ΔCT ubiquitination with WT or lysine-less ubiquitin. Ubiquitination reactions were performed as described in Experimental procedures with STAM1 variant ΔC-tail in which lysine 136 has been substituted with an arginine (K136R). Reactions were performed in the presence of 40 nM preactivated β-arrestin1 and with WT ubiquitin (WT Ub) (A) or lysine-less ubiquitin (0K Ub) (D). Reactions were performed with or without 1 mM ATP and terminated immediately (0 min) or after incubation at 37 C for 60 min in 2× sample buffer. Reactions were analyzed by 7% SDS-PAGE and immunoblotting with the indicated antibodies. Polyubiquitinated [Ub(n)], unmodified and monoubiquitinated (mono-Ub) STAM1 are indicated. Representative immunoblots are shown. STAM1 ubiquitination was quantified from the STAM (B and E) and ubiquitin (C and F) immunoblots using densitometry and shown as bar graphs. Data were expressed relative to the signal from WT ΔCT and represent the mean ± S.D. from three independent experiments. Data were analyzed by ene-way ANOVA GraphPad Prism. Adjusted p values are indicated. STAM, signal-transducing adaptor molecule.

Article Snippet: UbcH7 (E2; cat# E2-640–100), lysine-less ubiquitin (0K-Ub; cat# UM-NOK) and lysine-less biotinylated ubiquitin (0K-Ub biotin; cat# UB-560) were from R&D Systems.

Techniques: Ubiquitin Proteomics, Variant Assay, Incubation, SDS Page, Western Blot

Journal: The Journal of biological chemistry

Article Title: β-arrestin1 is an E3 ubiquitin ligase adaptor for substrate linear polyubiquitination.

doi: 10.1016/j.jbc.2023.105474

Figure Lengend Snippet: Figure 6. Evaluation of deleting the C-terminus on STAM1 ubiquitination. Ubiquitination reactions as described in Experimental procedures were performed with a truncated variant of STAM1 (ΔC-tail) that is still able to bind to β-arr1. Reactions were incubated with 40 nM WT β-arr1 (WT) or pre- activated variant of β-arr1 (R169E) or vehicle (n/a) in the presence or absence of 1 mM ATP with WT ubiquitin (WT Ub) (A) or lysine-less ubiquitin (0K Ub) (D). Reactions were terminated in 2× sample buffer immediately (0 min) or after incubation at 37 C for 60 min. Reactions were analyzed by 7% SDS-PAGE and immunoblotting. Polyubiquitinated [Ub(n)], unmodified, monoubiquitinated (mono-Ub) STAM1 and nonspecific bands are indicated. Representative im- munoblots are shown. STAM1 ubiquitination was quantified from the STAM (B and E) and ubiquitin (C and F) immunoblots using densitometry and shown as bar graphs. Data were expressed relative to the signal without βarr1 (“−“, minus symbol) and represent the mean ± S.D. from three independent ex- periments. Data were analyzed by one-way ANOVA GraphPad Prism. Adjusted p values are indicated. STAM, signal-transducing adaptor molecule.

Article Snippet: UbcH7 (E2; cat# E2-640–100), lysine-less ubiquitin (0K-Ub; cat# UM-NOK) and lysine-less biotinylated ubiquitin (0K-Ub biotin; cat# UB-560) were from R&D Systems.

Techniques: Ubiquitin Proteomics, Variant Assay, Incubation, SDS Page, Western Blot

Journal: Nucleic Acids Research

Article Title: A ubiquitin switch controls autocatalytic inactivation of the DNA–protein crosslink repair protease SPRTN

doi: 10.1093/nar/gkaa1224

Figure Lengend Snippet: Promiscuous E3-independent monoubiquitylation of SPRTN’s C-terminal tail. ( A–D ) Monoubiquitylation status of truncated SPRTN variants. Plasmids encoding tagged full-length (FL) SPRTN or truncations (carrying the indicated lysine to arginine (KR) substitutions or the UBZ* variant, D473A) were transiently transfected in HeLa T-REx Flp-In cells. Expression of SPRTN was induced by addition of doxycycline for 6 h prior to cell lysis (including a co-treatment with a ubiquitin-activating enzyme E1 inhibitor (E1i) as indicated) and analysed by SDS-PAGE and western blotting. ( E ) In vitro ubiquitylation assays containing SPRTN-EQ (410 nM), UBE2D3 (4 μM), E1 ubiquitin activating enzyme (300 nM), ubiquitin (50 μM) and ATP (2 mM) as indicated were incubated for 1.5 h at 30°C. Reactions were stopped by addition of LDS sample buffer and subjected to SDS-PAGE followed by staining with InstantBlue Coomassie protein stain. ( F ) In vitro ubiquitylation assays containing SPRTN-EQ or SPRTN-EQ-UBZ* (410 nM), E1 ubiquitin activating enzyme (300 nM), ubiquitin (50 μM), ATP (2 mM), UBE2D3 as indicated (8 μM) and increasing amounts of the catalytic domain of USP2 (USP2 cd ) (0, 250 or 500 nM) were incubated for 1.5 h at 30°C. Reactions were stopped by addition of LDS sample buffer and subjected to SDS-PAGE followed by staining with InstantBlue Coomassie protein stain.

Article Snippet: In brief, human E2 ubiquitin conjugating enzymes (2 μM) were incubated together with catalytically inactive SPRTN-E112Q (EQ) (2 μM), E1 ubiquitin activating enzyme (100 nM), ubiquitin (R&D Systems, U-100H, 50 μM) or no lysines N-Terminal Biotin ubiquitin (R&D Systems, UB-NOK-050, 50 μM), DNA (11.1 nM ФX174 virion) and ATP (2 mM) for 1.5 h at 30°C.

Techniques: Variant Assay, Transfection, Expressing, Lysis, Ubiquitin Proteomics, SDS Page, Western Blot, In Vitro, Incubation, Staining

Journal: Nucleic Acids Research

Article Title: A ubiquitin switch controls autocatalytic inactivation of the DNA–protein crosslink repair protease SPRTN

doi: 10.1093/nar/gkaa1224

Figure Lengend Snippet: Monoubiquitylation promotes SPRTN degradation and autocleavage. ( A ) Stability of endogenous SPRTN was determined with a cycloheximide-chase experiment in HeLa-T-REx Flp-In cells. Cells were incubated with cycloheximide for the indicated amount of time (with or without a 2-h pre-treatment with the proteasome inhibitor MG132) prior to cell lysis and analysis by western blotting. ( B ) Polyubiquitylation of stably expressed doxycycline-inducible YFP-SPRTN-Strep or of YFP-SPRTN-UBZ*-Strep was determined in HeLa-T-REx Flp-In cells upon treatment with proteasome inhibitor MG132 for the indicated amount of time prior to cell lysis and analysis by western blotting. ( C ) Stability of stably expressed doxycycline-inducible YFP-SPRTN-Strep or a linear SPRTN-Ubiquitin fusion (YFP-SPRTN-Ub LF ) was determined in HeLa-T-REx Flp-In cells using a cycloheximide-chase experiment. Cells were incubated in the presence of cycloheximide for the indicated amount of time (with or without a 2-h pre-treatment with the proteasome inhibitor MG132) prior to cell lysis and analysis by western blotting. ( D ) Indicated YFP-SPRTN-Strep or linear SPRTN-Ubiquitin fusion (YFP-SPRTN-Ub LF ) variants were transiently transfected in HeLa-T-REx Flp-In cells. SPRTN autocleavage fragments were enriched on GFP-trap resins, followed by western blotting against the N-terminal YFP-tag. Western blotting of cell lysates against GAPDH serves as loading control. Asterisks indicate autocleavage fragments. ( E ) Indicated YFP-SPRTN-Strep variants were transiently transfected in HeLa-T-REx Flp-In cells in combination with Flag-tagged full-length USP7 (WT or the catalytically inactive CS variant) or the empty vector. SPRTN autocleavage fragments were enriched on GFP-trap resins, followed by western blotting against the N-terminal YFP-tag. Western blotting against GAPDH of cell lysates serves as loading control. Asterisks indicate autocleavage fragments. ( F ) HAP1 cells were treated with increasing amounts of formaldehyde (FA, 0.25, 0.5, 1 and 2 mM) for 2 h (either with or without a 2-h pre-treatment with ubiquitin-activating enzyme E1 inhibitor as indicated) prior to cell lysis and analysis by western blotting. Asterisks indicate autocleavage fragments. ( G ) HeLa-T-REx Flp-In cells were treated with proteasome inhibitor MG132 for the indicated amount of time prior to cell lysis and analysis by western blotting. Asterisks indicate autocleavage fragments.

Article Snippet: In brief, human E2 ubiquitin conjugating enzymes (2 μM) were incubated together with catalytically inactive SPRTN-E112Q (EQ) (2 μM), E1 ubiquitin activating enzyme (100 nM), ubiquitin (R&D Systems, U-100H, 50 μM) or no lysines N-Terminal Biotin ubiquitin (R&D Systems, UB-NOK-050, 50 μM), DNA (11.1 nM ФX174 virion) and ATP (2 mM) for 1.5 h at 30°C.

Techniques: Incubation, Lysis, Western Blot, Stable Transfection, Ubiquitin Proteomics, Transfection, Control, Variant Assay, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A ubiquitin switch controls autocatalytic inactivation of the DNA–protein crosslink repair protease SPRTN

doi: 10.1093/nar/gkaa1224

Figure Lengend Snippet: Monoubiquitylation promotes SPRTN autocleavage in trans . ( A ) Recombinant SPRTN or a linear SPRTN-Ubiquitin fusion (SPRTN-Ub LF ) (500 nM) were incubated with histone H1 alone or in the presence of either single- (ss) Virion or double-stranded (ds) RFI ФX174 DNA (11.1 nM) for 60 min at 25°C. Salt concentrations were as indicated. Reactions were analysed by SDS-PAGE followed by western blotting and staining with InstantBlue Coomassie protein stain. Quantification of western blots of results of SPRTN and histone H1 cleavage: values represent the mean ± SD of four independent experiments. ( B ) Indicated model protein G-oligonucleotide conjugates (25nM) were incubated alone or in the presence of recombinant SPRTN (6.25 nM, WT or a linear SPRTN-Ubiquitin fusion (SPRTN-Ub LF )) for 2 h at 25°C prior to separation by native PAGE. Right panel, quantification of DPC cleavage: values represent the mean ± SD of three independent experiments. ( C ) Recombinant catalytically inactive Flag-SPRTN-EQ (500 nM) was incubated alone or in combination with active SPRTN (500 nM, WT or a linear SPRTN-Ubiquitin fusion (SPRTN-Ub LF )) in the presence of DNA (ФX174 RFI dsDNA, 11.1 nM) for 60 min at 25°C. Salt concentrations were as indicated. Reactions were subjected to SDS-PAGE followed by staining with InstantBlue Coomassie protein stain and western blotting.

Article Snippet: In brief, human E2 ubiquitin conjugating enzymes (2 μM) were incubated together with catalytically inactive SPRTN-E112Q (EQ) (2 μM), E1 ubiquitin activating enzyme (100 nM), ubiquitin (R&D Systems, U-100H, 50 μM) or no lysines N-Terminal Biotin ubiquitin (R&D Systems, UB-NOK-050, 50 μM), DNA (11.1 nM ФX174 virion) and ATP (2 mM) for 1.5 h at 30°C.

Techniques: Recombinant, Ubiquitin Proteomics, Incubation, SDS Page, Western Blot, Staining, Clear Native PAGE

Journal: Nucleic Acids Research

Article Title: A ubiquitin switch controls autocatalytic inactivation of the DNA–protein crosslink repair protease SPRTN

doi: 10.1093/nar/gkaa1224

Figure Lengend Snippet: Regulation of SPRTN by monoubiquitylation and USP7. Proposed model for the regulation of SPRTN by monoubiquitylation and USP7-mediated deubiquitylation. SPRTN is subjected to constitutive promiscuous monoubiquitylation of its C-terminal tail. The modification is shielded by SPRTN’s ubiquitin binding zinc-finger (UBZ). Monoubiquitylation affects SPRTN twofold. It primes SPRTN in cis for proteasomal degradation by inducing polyubiquitylation while also triggering inactivation by fostering autocleavage of other SPRTN molecules in trans . USP7 relieves this inhibition by deubiquitylating SPRTN upon induction of DNA–protein crosslinks (DPCs).

Article Snippet: In brief, human E2 ubiquitin conjugating enzymes (2 μM) were incubated together with catalytically inactive SPRTN-E112Q (EQ) (2 μM), E1 ubiquitin activating enzyme (100 nM), ubiquitin (R&D Systems, U-100H, 50 μM) or no lysines N-Terminal Biotin ubiquitin (R&D Systems, UB-NOK-050, 50 μM), DNA (11.1 nM ФX174 virion) and ATP (2 mM) for 1.5 h at 30°C.

Techniques: Modification, Ubiquitin Proteomics, Binding Assay, Inhibition