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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA-protein crosslinks (DPCs) pose a threat to genome stability.
• The protease SPRTN is involved in DPC degradation.
• A functional link between SPRTN and the protein unfoldase p97 (VCP/Cdc48) was suggested but not directly demonstrated.

Purpose of the Study:

• To biochemically demonstrate the functional cooperation between p97 and SPRTN in degrading DPCs.
• To elucidate the mechanism by which p97 assists SPRTN in processing DPCs.
• To explore the potential of targeting p97 in cancer therapy.

Main Methods:

• Biochemical reconstitution using purified proteins.
• In vitro assays to assess protein unfolding and proteolysis.
• Use of a DNA-crosslinked fluorescent reporter protein (Eos-DPC).
• Investigation of the role of p97's ubiquitin adapter, Ufd1-Npl4.

Main Results:

• Purified SPRTN alone could not degrade tightly-folded DNA-crosslinked proteins.
• p97, with its adapter Ufd1-Npl4, unfolded ubiquitinated Eos-DPC, preparing it for proteolysis.
• SPRTN, in cooperation with p97/Ufd1-Npl4, proteolyzed the unfolded DPC, recognizing the DNA-crosslink.
• p97 demonstrated the ability to unfold protein substrates despite the presence of DNA crosslink sites.

Conclusions:

• p97 and Ufd1-Npl4 cooperate to facilitate SPRTN-mediated proteolysis of tightly-folded DPCs.
• This mechanism involves p97 unfolding the DPC, allowing SPRTN access to the crosslinked site.
• These findings are crucial for understanding cellular DPC management and developing combination cancer therapies targeting p97.