A ubiquitin switch controls autocatalytic inactivation of the DNA-protein crosslink repair protease SPRTN

Abstract

Repair of covalent DNA-protein crosslinks (DPCs) by the metalloprotease SPRTN prevents genome instability, premature aging and carcinogenesis. SPRTN is specifically activated by DNA structures containing single- and double-stranded features, but degrades the protein components of DPCs promiscuously and independent of amino acid sequence. This lack of specificity is useful to target diverse protein adducts, however, it requires tight control in return, in order to prohibit uncontrolled proteolysis of chromatin proteins. Here, we discover the components and principles of a ubiquitin switch, which negatively regulates SPRTN. We demonstrate that monoubiquitylation is induced in an E3 ligase-independent manner and, in contrast to previous assumptions, does not control chromatin access of the enzyme. Data obtained in cells and in vitro reveal that monoubiquitylation induces inactivation of the enzyme by triggering autocatalytic cleavage in trans while also priming SPRTN for proteasomal degradation in cis. Finally, we show that the deubiquitylating enzyme USP7 antagonizes this negative control of SPRTN in the presence of DPCs.

Figure 1.

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.

Figure 2.

An in vitro screen identifies DUBs targeting ubiquitylated SPRTN. (A) Schematic depiction of the screening strategy employed to test seventy-one human deubiquitylating enzymes (DUBs) for their ability to deubiquitylate SPRTN. (B) Deconvolution of positive pools identified in Supplementary Figure S2A reveals four candidate DUBs. Lysates prepared from fifteen clones present in the five positive pools were incubated for 30 min at 25°C together with purified partially-monoubiquitylated YFP-SPRTN-EQ-Strep. Reactions were stopped by addition of LDS sample buffer and analysed by SDS-PAGE and western blotting using anti-Strep antibody. Lysates of BL21 cells served as negative control, the unspecific deubiquitylating activity of the catalytic domain of USP2 (USP2^cd) as positive control.

Figure 3.

USP7 interacts with and targets ubiquitylated SPRTN in human cells. (A) Analysis of DUB overexpression-induced deubiquitylation of endogenous SPRTN in HeLa T-REx Flp-In cells. Indicated N-terminally Flag-tagged DUBs were transiently expressed for one day before cells were lysed and analysed by western blotting. (B) Analysis of DUB overexpression-induced deubiquitylation of doxycycline-inducible YFP-SPRTN-Strep stably expressed in HeLa T-REx Flp-In cells. Indicated N-terminally Flag-tagged DUBs were transiently expressed for one day before cells were lysed and analysed by western blotting. (C) Increasing amounts of N-terminally Flag-tagged USP7 (or the catalytically inactive CS variant) were transiently expressed in HeLa T-REx Flp-In cells for one day before cells were lysed and analysed by western blotting. (D) Increasing amounts of N-terminally Flag-tagged USP7 (or the catalytically inactive CS variant) were transiently expressed in HeLa T-REx Flp-In cells stably expressing doxycycline-inducible YFP-SPRTN-Strep for one day before cells were lysed and analysed by western blotting. (E) Plasmids encoding Flag-tagged full-length USP7 (WT or the catalytically inactive CS variant) were transiently transfected in HeLa T-REx Flp-In cells stably expressing the indicated doxycycline-inducible YFP-SPRTN-Strep variants. Binding was analysed by co-immunoprecipitation using anti-Flag beads followed by western blotting. (F) Schematic depiction of USP7’s domain structure and protein truncations used for co-immunoprecipitation analysis with SPRTN (upper panel). Plasmids encoding Flag-tagged full-length USP7 (WT or the catalytically inactive CS variant) or the respective truncations were transiently transfected in HeLa T-REx Flp-In cells stably expressing doxycycline-inducible YFP-SPRTN-Strep. Binding was analysed by co-immunoprecipitation using anti-Flag beads followed by western blotting (lower panel).

Figure 4.

USP7 deubiquitylates SPRTN upon DPC induction. (A) HCT116 WT or USP7 knock-out (KO) cells were treated with 2 mM formaldehyde (FA) for 2 h. Cells were either lysed directly in LDS sample buffer (total) or subjected to chromatin fractionation. Samples were then analyzed by SDS-PAGE followed by western blotting. Asterisks indicates a cross-reactive band. (B) Clonal DLD1 USP7 KO cells and matched WT control cells were treated with 2 mM formaldehyde (FA) for 3 h. Cells were either lysed directly in LDS sample buffer (total) or subjected to chromatin fractionation. Samples were then analysed by SDS-PAGE followed by western blotting. Asterisks indicates a cross-reactive band. (C) Clonal HAP1 USP7 KO cells and matched WT control cells were treated with 2 mM formaldehyde (FA) for 2 h. Cells were either lysed directly in LDS sample buffer (total) or subjected to chromatin fractionation. Samples were then analysed by SDS-PAGE followed by western blotting. Asterisks indicates a cross-reactive band. (D) Clonal DLD1 USP7 KO cells and matched WT control cells were treated with the indicated formaldehyde concentrations for 2 h. After 48 h cell viability was determined using the alamarBlue cell viability assay. Values represent the mean ± SD of three technical replicates normalized to the mean of untreated controls of each cell line (E) Clonal HAP1 USP7 KO cells and matched WT control cells were treated with the indicated formaldehyde concentrations for 72 h. Cell viability was then determined using the alamarBlue cell viability assay. Values represent the mean ± SD of three technical replicates normalized to the mean of untreated controls of each cell line. (F) YFP-tagged full-length USP7 (WT or the catalytically inactive CS variant) or the empty vector were transiently transfected in DLD1 USP7 KO cells and matched WT control cells. Cells were treated with 1 mM formaldehyde for 2 h. After 48 h cell viability was determined using the alamarBlue cell viability assay. Values represent the mean ± SEM of four independent biological replicates normalized to the mean of untreated controls of each cell line. Significance was determined using a paired t-test (*P-value < 0.05). (G) Cellular DPCs were quantified in clonal HAP1 USP7 KO cells and matched WT control cells treated with 75 μM formaldehyde for 2 h using a KCl/SDS precipitation assay. DPCs were measured as the ratio of crosslinked DNA compared to total DNA. Values represent the mean ± SD of three technical replicates.

Figure 5.

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.

Figure 6.

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.

Figure 7.

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).