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Peng H , Cassel J , McCracken DS , Prokop JW , Sementino E , Cheung M , Collop PR , Polo A , Joshi S , Mandell JP , Ayyanathan K , Hinds D , Malkowicz SB , Harbour JW , Bowcock AM , Salvino J , Kennedy EJ , Testa JR , Rauscher FJ
Kinetic Characterization of ASXL1/2-Mediated Allosteric Regulation of the BAP1 Deubiquitinase
Mol Cancer Res. 2021 Mar 17
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BAP1 is an ubiquitin hydrolase whose deubiquitinase activity is mediated by polycomb group-like protein ASXL2. Cancer-related BAP1 mutations/deletions lead to loss-of-function by targeting the catalytic (UCH) or ULD domains of BAP1, and the latter disrupts binding to ASXL2, an obligate partner for BAP1 enzymatic activity. However, the biochemical and biophysical properties of domains involved in forming the enzymatically active complex are unknown. Here, we report the molecular dynamics, kinetics and stoichiometry of these interactions. We demonstrate that interactions between BAP1 and ASXL2 are direct, specific, and stable to biochemical and biophysical manipulations as detected by isothermal titration calorimetry, GST association, and optical biosensor assays. Association of the ASXL2-AB box greatly stimulates BAP1 activity. A stable ternary complex is formed, comprised of the BAP1-UCH, BAP1-ULD, and ASXL2-AB domains. Stoichiometric analysis revealed that one molecule of the ULD domain directly interacts with one molecule of the AB box. Real-time kinetics analysis of the ULD/AB protein complex to the BAP1 UCH domain, based on SPR, indicated that formation of the ULD/AB complex with the UCH domain is a single-step event with fast association and slow dissociation rates. In vitro experiments validated in cells that ASXL-AB box directly regulates BAP1 activity. Implications: Collectively, these data elucidate molecular interactions between specific protein domains regulating BAP1 deubiquitinase activity, thus establishing a foundation for small-molecule approaches to reactivate latent wild-type BAP1 catalytic activity in BAP1-mutant cancers.
1557-3125 Peng, Hongzhuang Cassel, Joel McCracken, Daniel S Prokop, Jeremy W Sementino, Eleonora Cheung, Mitchell Collop, Paul R Polo, Alexander Joshi, Surbhi Mandell, Jacob P Ayyanathan, Kasirajan Hinds, David Malkowicz, S Bruce Harbour, J William Orcid: 0000-0002-1104-9809 Bowcock, Anne M Orcid: 0000-0001-8691-9090 Salvino, Joseph Orcid: 0000-0002-2184-5980 Kennedy, Eileen J Orcid: 0000-0001-5610-1677 Testa, Joseph R Orcid: 0000-0003-1301-5175 Rauscher, Frank J Journal Article United States Mol Cancer Res. 2021 Mar 17:molcanres.0080.2020. doi: 10.1158/1541-7786.MCR-20-0080.