Ubiquitin-directed AAA+ ATPase p97/VCP unfolds stable proteins crosslinked to DNA for proteolysis by SPRTN

Abstract

The protease SPRTN degrades DNA-protein crosslinks (DPCs) that threaten genome stability. SPRTN has been connected to the ubiquitin-directed protein unfoldase p97 (also called VCP or Cdc48), but a functional cooperation has not been demonstrated directly. Here, we biochemically reconstituted p97-assisted proteolysis with purified proteins and showed that p97 targets ubiquitin-modified DPCs and unfolds them to prepare them for proteolysis by SPRTN. We demonstrate that purified SPRTN alone was unable to degrade a tightly-folded Eos fluorescent reporter protein even when Eos was crosslinked to DNA (Eos-DPC). However, when present, p97 unfolded poly-ubiquitinated Eos-DPC in a manner requiring its ubiquitin adapter, Ufd1-Npl4. Notably, we show that, in cooperation with p97 and Ufd1-Npl4, SPRTN proteolyzed unfolded Eos-DPC, which relied on recognition of the DNA-crosslink by SPRTN. In a simplified unfolding assay, we further demonstrate that p97, while unfolding a protein substrate, can surmount the obstacle of a DNA crosslink site in the substrate. Thus, our data demonstrate that p97, in conjunction with Ufd1-Npl4, assists SPRTN-mediated proteolysis of tightly-folded proteins crosslinked to DNA, even threading bulky protein-DNA adducts. These findings will be relevant for understanding how cells handle DPCs to ensure genome stability and for designing strategies that target p97 in combination cancer therapy.

Keywords: AAA+ ATPase; Cdc48; DNA-protein crosslink; SPRTN; VCP; Wss1; p97; protein unfolding.

Figure 1

Generation of a poly-ubiquitylated DNA-protein-crosslink reporter substrate protein.A, steps of the substrate reporter generation. A di-ubiquitin-Eos fusion (Ub2-Eos) was generated in bacteria. Ub2-Eos was first irradiated with UV light to introduce a backbone break in Eos in roughly 30% of the protein population, which entails a conversion from the green to the red fluorescent form of Eos and facilitates monitoring unfolding reactions. A double-strand DNA oligonucleotide with a 5′ overhang was crosslinked to a cysteine in the Eos moiety, yielding Ub2-Eos-DPC. The oligonucleotide was coupled to Dy781 to facilitate visualization of the DPC and its fragments in SDS-PAGE. The reporter substrate was subsequently polyubiquitylated on the N-terminal ubiquitin moieties yielding Ubn-Eos-DPC. B, the steps described in (A) analyzed in SDS-PAGE and visualized by Coomassie staining (left) and infrared Dy781 fluorescence scan (right). Note that N- and C-terminal Eos fragments remain associated in the native protein but are separated in the denaturing gel. C, Coomassie gel of purified protein components. DPC, DNA-protein crosslink.

Figure 2

Proteolysis of the DPC substrate by SPRTN is dependent on p97- and Ufd1-Npl4–mediated unfolding.A, Ufd1-Npl4 and SPRTN bind p97 at the same time. Binding assay containing Strep-tagged SPRTN, p97, and increasing concentrations of Ufd1-Npl4 followed by pull-down of SPRTN. Proteins were visualized by Western blot. Note that Ufd1-Npl4 did not compete with SPRTN for p97, but instead that Ufd1-Npl4 coisolated with SPRTN in the presence of p97. B, the Ubn-Eos-DPC substrate was incubated with p97 and Ufd1-Npl4, and without or with SPRTN for 0 to 30 min at 37 °C, as indicated. ATP was added as indicated to support p97 unfolding activity. Samples were separated by SDS-PAGE followed by a fluorescence scan detecting Dy781. Note that while generation of the ∼40 kDa Eos-containing fragment is ATP independent, the generation of an internal ∼20 kDa Eos proteolysis fragment requires ATP-driven p97 activity. C, quantification of the chromophore intensity associated with the ∼20 kDa band in (B) using a fluorescence scanner. n = 3; the error bars represent mean ± s.d. One-way ANOVA, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001. D, assay as in (A). Individual components were omitted or p97 activity was inhibited by CB5083 or NMS-873, as indicated. Note that generation of the ∼20 kDa proteolysis product consistently requires p97 activity. E, quantification of the chromophore intensity associated with the ∼20 kDa band in (D) using a fluorescence scanner. Values were normalized to signal in full reaction condition. n = 3; the error bars represent mean ± s.d. One-way ANOVA, ∗∗p < 0.01. DPC, DNA-protein crosslink; SPRTN, SprT-like N-terminal domain.

Figure 3

Real-time monitoring of DPC substrate unfolding and proteolysis by p97-Ufd1-Npl4 and SPRTN.A, depiction of the Eos fluorescence-based substrate unfolding assay. Green (unbroken) and red (broken) Ubn-Eos-DPC are recruited to p97 by Ufd1-Npl4. Unfolding of red Eos leads to net loss of Eos fluorescence due to the backbone break that prevents refolding. Unfolding of green Eos results only in an initial loss of fluorescence that plateaus due to refolding of the reporter. Additional loss of fluorescence therefore reports on proteolysis of the reporter. B, assay as described in (A). Green and red Eos fluorescence was monitored over time in the presence and absence of SPRTN, as indicated. Note that loss of red fluorescence is not affected by SPRTN, as expected. Instead, loss of green fluorescence is accelerated indicating proteolysis of the reporter by SPRTN following p97-mediated unfolding. n = 3; mean ± s.e.m. DPC, DNA-protein crosslink; SPRTN, SprT-like N-terminal domain.

Figure 4

p97-assisted DPC reporter proteolysis depends on DNA binding by SPRTN and is stimulated by SPRTN-p97 interaction.A, Coomassie gels of indicated SPRTN mutant proteins used. Asterisks indicate impurities. B, domain structure of WT SPRTN and mutants. E112Q mutation abolishes protease activity. Red asterisk indicates mutations. C, DPC reporter proteolysis assay as in Figure 2A with WT SPRTN and indicated mutants. D, quantification of chromophore intensity associated with the ∼20 kDa band in (C) using a fluorescence scanner. n = 3; the error bars represent mean ± s.d. One-way ANOVA, ∗p < 0.05; ∗∗∗∗p < 0.0001. E, mutation of the SHP box in SPRTN abolishes p97 binding. p97 was incubated with WT SPRTN or SPRTN with mutated SHP box (ShpMut) before immunoprecipitation of p97. p97 and coimmunoprecipitated SPRTN was detected by Western blot. BR, basic region; DPC, DNA-protein crosslink; SHP, suppressor of high copy PP1; SPRTN, SprT-like N-terminal domain; UBZ, ubiquitin-binding zinc-finger; ZBD, zinc-binding domain.

Figure 5

p97 can surmount DNA crosslink sites.A, an alternative, ubiquitin-independent p97-mediated unfolding reaction with defined unfolding initiation site relative to the Eos unfolding reporter moiety. A protein complex of PP1γ catalytic subunit, SDS22 and Inhibitor-3 (I3), is disassembled by threading of I3 through the p97 channel. Unfolding is initiated at I3 by binding to the p37 adapter and progresses to the Eos fluorescent unfolding sensor. B, a reporter substrate was generated with a SNAP-tag between the I3 and Eos moieties and purified with PP1 γ (P) and SDS22 (S) as a complex (SP–Eos-Snap-I3). A 30 nt DNA oligonucleotide was conjugated to the SNAP-tag. Coomassie-stained SDS-PAGE of free and DNA-conjugated SP–Eos-Snap-I3. C, unfolding assay with SP–Eos-Snap-I3 substrate conjugated to DNA or not, as indicated, p97 and the I3-specific adapter p37. p37 was omitted as a negative control (-p37). Note that unfolding kinetics are similar irrespective of the conjugated DNA. I3, inhibitor 3; PP1, protein phosphatase-1 catalytic subunit.

Figure 6

Model for p97-assisted DPC proteolysis by SPRTN. Loosely folded DPCs can be directly accessed by SPRTN, resulting in proteolysis of the DPC allowing subsequent removal of the remaining fragment (upper panel). More tightly folded DPCs require ubiquitylation and assistance by p97. p97 is recruited by Ufd1-Npl4 to the ubiquitylated DPC and unfolds the crosslinked protein to allow proteolysis by SPRTN (lower panel). DPC, DNA-protein crosslink; SPRTN, SprT-like N-terminal domain.