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