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.
Subject(s)
DNA-Binding Proteins/metabolism , Proteins , Ubiquitin , Valosin Containing Protein , ATPases Associated with Diverse Cellular Activities/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA/metabolism , Genomic Instability , Humans , Proteins/metabolism , Proteolysis , Ubiquitin/metabolism , Valosin Containing Protein/genetics , Valosin Containing Protein/metabolismABSTRACT
The AAA-ATPase VCP/p97 cooperates with the SEP-domain adapters p37, UBXN2A and p47 in stripping inhibitor-3 (I3) from protein phosphatase-1 (PP1) for activation. In contrast to p97-mediated degradative processes, PP1 complex disassembly is ubiquitin-independent. It is therefore unclear how selective targeting is achieved. Using biochemical reconstitution and crosslink mass spectrometry, we show here that SEP-domain adapters use a multivalent substrate recognition strategy. An N-terminal sequence element predicted to form a helix, together with the SEP-domain, binds and engages the direct target I3 in the central pore of p97 for unfolding, while its partner PP1 is held by a linker between SHP box and UBX domain locked onto the peripheral N-domain of p97. Although the I3-binding element is functional in p47, p47 in vitro requires a transplant of the PP1-binding linker from p37 for activity stressing that both sites are essential to control specificity. Of note, unfolding is then governed by an inhibitory segment in the N-terminal region of p47, suggesting a regulatory function. Together, this study reveals how p97 adapters engage a protein complex for ubiquitin-independent disassembly while ensuring selectivity for one subunit.
Subject(s)
Adenosine Triphosphatases/chemistry , Multiprotein Complexes/chemistry , Nuclear Proteins/chemistry , Protein Conformation , Protein Phosphatase 1/chemistry , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/ultrastructure , Amino Acid Sequence/genetics , Catalytic Domain/genetics , Crystallography, X-Ray , Humans , Metalloendopeptidases/chemistry , Metalloendopeptidases/genetics , Multiprotein Complexes/genetics , Multiprotein Complexes/ultrastructure , Nuclear Proteins/genetics , Nuclear Proteins/ultrastructure , Protein Binding/genetics , Protein Phosphatase 1/genetics , Protein Phosphatase 1/ultrastructure , Protein Structure, Tertiary , Protein Subunits/chemistry , Ubiquitin/genetics , Ubiquitins/chemistry , Ubiquitins/geneticsABSTRACT
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
ABSTRACT
Urothelial carcinoma (UC), the most common cancer of the urinary bladder causes severe morbidity and mortality, e.g. about 40.000 deaths in the EU annually, and incurs considerable costs for the health system due to the need for prolonged treatments and long-term monitoring. Extensive aberrant DNA methylation is described to prevail in urothelial carcinoma and is thought to contribute to genetic instability, altered gene expression and tumor progression. However, it is unknown how this epigenetic alteration arises during carcinogenesis. Intact methyl group metabolism is required to ensure maintenance of cell-type specific methylomes and thereby genetic integrity and proper cellular function. Here, using two independent techniques for detecting DNA methylation, we observed DNA hypermethylation of the 5'-regulatory regions of the key methyl group metabolism genes ODC1, AHCY and MTHFR in early urothelial carcinoma. These hypermethylation events are associated with genome-wide DNA hypomethylation which is commonly associated with genetic instability. We therefore infer that hypermethylation of methyl group metabolism genes acts in a feed-forward cycle to promote additional DNA methylation changes and suggest a new hypothesis on the molecular etiology of urothelial carcinoma.
