Kinetic Characterization of ASXL1/2-Mediated Allosteric Regulation of the BAP1 Deubiquitinase
©2021 American Association for Cancer Research..
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 ubiquitin C-terminal hydrolase (UCH) or UCH37-like domain (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 (ITC), 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 kinetic analysis of the ULD/AB protein complex to the BAP1-UCH domain, based on surface plasmon resonance, 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 the 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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:19 |
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| Enthalten in: |
Molecular cancer research : MCR - 19(2021), 7 vom: 01. Juli, Seite 1099-1112 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Peng, Hongzhuang [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 24.01.2022 Date Revised 24.01.2022 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1158/1541-7786.MCR-20-0080 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 100 | 1 | |a Peng, Hongzhuang |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Kinetic Characterization of ASXL1/2-Mediated Allosteric Regulation of the BAP1 Deubiquitinase |
| 264 | 1 | |c 2021 | |
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| 500 | |a Date Revised 24.01.2022 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a ©2021 American Association for Cancer Research. | ||
| 520 | |a 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 ubiquitin C-terminal hydrolase (UCH) or UCH37-like domain (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 (ITC), 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 kinetic analysis of the ULD/AB protein complex to the BAP1-UCH domain, based on surface plasmon resonance, 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 the 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a Research Support, U.S. Gov't, Non-P.H.S. | |
| 650 | 7 | |a ASXL1 protein, human |2 NLM | |
| 650 | 7 | |a ASXL2 protein, human |2 NLM | |
| 650 | 7 | |a BAP1 protein, human |2 NLM | |
| 650 | 7 | |a Repressor Proteins |2 NLM | |
| 650 | 7 | |a Tumor Suppressor Proteins |2 NLM | |
| 650 | 7 | |a Ubiquitin |2 NLM | |
| 650 | 7 | |a Ubiquitin Thiolesterase |2 NLM | |
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| 700 | 1 | |a Cassel, Joel |e verfasserin |4 aut | |
| 700 | 1 | |a McCracken, Daniel S |e verfasserin |4 aut | |
| 700 | 1 | |a Prokop, Jeremy W |e verfasserin |4 aut | |
| 700 | 1 | |a Sementino, Eleonora |e verfasserin |4 aut | |
| 700 | 1 | |a Cheung, Mitchell |e verfasserin |4 aut | |
| 700 | 1 | |a Collop, Paul R |e verfasserin |4 aut | |
| 700 | 1 | |a Polo, Alexander |e verfasserin |4 aut | |
| 700 | 1 | |a Joshi, Surbhi |e verfasserin |4 aut | |
| 700 | 1 | |a Mandell, Jacob P |e verfasserin |4 aut | |
| 700 | 1 | |a Ayyanathan, Kasirajan |e verfasserin |4 aut | |
| 700 | 1 | |a Hinds, David |e verfasserin |4 aut | |
| 700 | 1 | |a Malkowicz, S Bruce |e verfasserin |4 aut | |
| 700 | 1 | |a Harbour, J William |e verfasserin |4 aut | |
| 700 | 1 | |a Bowcock, Anne M |e verfasserin |4 aut | |
| 700 | 1 | |a Salvino, Joseph |e verfasserin |4 aut | |
| 700 | 1 | |a Kennedy, Eileen J |e verfasserin |4 aut | |
| 700 | 1 | |a Testa, Joseph R |e verfasserin |4 aut | |
| 700 | 1 | |a Rauscher, Frank J |c 3rd |e verfasserin |4 aut | |
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