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Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
Published on: December 5, 2019
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BMI1-RING1B is an autoinhibited RING E3 ubiquitin ligase
Asad M Taherbhoy1, Oscar W Huang1, Andrea G Cochran1
1Department of Early Discovery Biochemistry, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA.
Nature Communications
|July 8, 2015
Summary
Polycomb repressive complex 1 (PRC1) E3 ligases, like BMI1/PCGF4, exhibit low enzymatic activity. This is offset by favorable nucleosome interactions, enabling efficient gene repression through histone H2A ubiquitination.
Area of Science:
- Epigenetics
- Molecular Biology
- Structural Biology
Background:
- Polycomb repressive complex 1 (PRC1) mediates histone H2A ubiquitination (H2Aub1), a key epigenetic mark for gene silencing during development.
- PRC1's E3 ligase activity is primarily attributed to the RING1A/B subunit, partnered with one of six PCGF proteins, including the oncogenic BMI1/PCGF4.
Purpose of the Study:
- To investigate the enzymatic activity of canonical PRC1 E3 ligases, particularly the PCGF4-RING1B complex.
- To elucidate the structural basis for the observed differences in E3 ligase activity between canonical and non-canonical PRC1 complexes.
- To understand how PRC1 achieves site-specific monoubiquitination of histone H2A.
Main Methods:
- Comparative analysis of E3 ligase activity between different PRC1 complexes.
- Structural determination of a high-activity PRC1 variant (PCGF5-RING1B) in complex with an E2 enzyme (UbcH5c).
- Site-directed mutagenesis to identify critical residues influencing ubiquitin transfer.
Main Results:
- Canonical PRC1 E3 ligases, such as PCGF4-RING1B, display intrinsically low enzymatic activity compared to non-canonical RING dimers.
- Structural analysis revealed subtle differences between PCGF4 and PCGF5 complexes, with two charged residues at the E2-ubiquitin interface being critical.
- In BMI1/PCGF4, these residues form a salt bridge potentially limiting ubiquitin transfer, while favorable nucleosome interactions compensate for low intrinsic activity, leading to efficient H2Aub1.
Conclusions:
- The intrinsic enzymatic activity of canonical PRC1 E3 ligases is modulated by specific residues at the E2-ubiquitin interface.
- Despite low intrinsic activity, PRC1 efficiently catalyzes H2Aub1 through favorable substrate interactions, underscoring the complexity of epigenetic regulation.
- Understanding these mechanisms provides insights into developmental gene regulation and potential therapeutic targets in cancer.
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