title: Mutant p97 exhibits species-specific changes of its ATPase activity and compromises the UBXD9-mediated monomerisation of p97 hexamers.
abstract: p97 (VCP) is a homo-hexameric triple-A ATPase that exerts a plethora of cellular processes. Heterozygous missense mutations of p97 cause at least five human neurodegenerative disorders. However, the specific molecular consequences of p97 mutations are hitherto widely unknown. Our in silico structural models of human and Dictyostelium p97 showed that the disease-causing human R93C, R155H, and R155C as well as Dictyostelium R154C, E219K, R154C/E219K p97 mutations constitute variations in surface-exposed locations. In-gel ATPase activity measurements of p97 monomers and hexamers revealed significant mutation- and species-specific differences. While all human p97 mutations led to an increase in ATPase activity, no changes could be detected for the Dictyostelium R154C mutant, which is orthologous to human R155C. The E219K mutation led to an almost complete loss of activity, which was partially recuperated in the R154C/E219K double-mutant indicating p97 inter-domain communication. By means of co-immunoprecipitation experiments we identified an UBX-domain containing Dictyostelium protein as a novel p97 interaction partner. We categorized all UBX-domain containing Dictyostelium proteins and named the interaction partner UBXD9. Pull-down assays and surface plasmon resonance analyses of Dictyostelium UBXD9 or the human orthologue TUG/ASPL/UBXD9 demonstrated direct interactions with p97 as well as species-, mutation- and ATP-dependent differences in the binding affinities. Sucrose density gradient assays revealed that both human and Dictyostelium UBXD9 proteins very efficiently disassembled wild-type, but to a lesser extent mutant p97 hexamers into monomers. Our results are consistent with a scenario in which p97 point mutations lead to differences in enzymatic activities and molecular interactions, which in the long-term result in a late-onset and progressive multisystem disease.
authors.affiliation: Centre for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany.
authors.forename: Ramesh
authors.initials: R
authors.lastname: Rijal
authors.affiliation: Centre for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany.
authors.forename: Khalid
authors.initials: K
authors.lastname: Arhzaouy
authors.affiliation: Centre for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany.
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Strucksberg
authors.affiliation: Structural Chemistry Program, Eskitis Institute, Griffith University, Brisbane, Queensland 4111, Australia.
authors.forename: Megan
authors.initials: M
authors.lastname: Cross
authors.affiliation: Structural Chemistry Program, Eskitis Institute, Griffith University, Brisbane, Queensland 4111, Australia; Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria 3030, Australia.
authors.forename: Andreas
authors.initials: A
authors.lastname: Hofmann
authors.affiliation: Institute of Neuropathology, University Hospital Erlangen, 91054 Erlangen, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Schröder
authors.affiliation: Centre for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany. Electronic address: christoph.clemen@uni-koeln.de.
authors.forename: Christoph S
authors.initials: CS
authors.lastname: Clemen
authors.affiliation: Centre for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany. Electronic address: ludwig.eichinger@uni-koeln.de.
authors.forename: Ludwig
authors.initials: L
authors.lastname: Eichinger
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008958:Models, Molecular; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000072417:Protein Domains; D013045:Species Specificity
chemical_list: D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D000251:Adenosine Triphosphatases; C404942:p97 ATPase
keywords: ALS; Dictyostelium discoideum; HMSN2; HSP; Hexamer-monomer disassembly; IBMPFD; Parkinson; Surface plasmon resonance; UBX domain containing protein UBXD9; UBX4/TUG/PUX/ASPL/ASPSCR1; p97/VCP/CDC48/TER/VAT ATPase