A bidentate Polycomb Repressive-Deubiquitinase complex is required for efficient activity on nucleosomes

Foglizzo, Martina; Middleton, Adam J.; Burgess, Abigail E.; Crowther, Jennifer M.; Dobson, Renwick C. J.; Murphy, James M.; Day, Catherine L.; Mace, Peter D.

doi:10.1038/s41467-018-06186-1

Cited by 32 publications

(57 citation statements)

References 69 publications

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“…During the revision of this manuscript, Foglizzo et al (2018) reported the crystal structure of a DmPR-DUB complex that closely resembles our complex. Based on their analyses the authors suggest a 2:2 complex where two Calypso/ASX heterodimers dimerize via the Calypso coiled-coil regions.…”

Section: Discussionmentioning

confidence: 74%

Structural Basis for the Activation of the Deubiquitinase Calypso by the Polycomb Protein ASX

Chittock

Grötsch

et al. 2019

Structure

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Section: Discussionmentioning

confidence: 74%

Structural Basis for the Activation of the Deubiquitinase Calypso by the Polycomb Protein ASX

Chittock

Grötsch

et al. 2019

Structure

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“…The stoichiometry of Calypso/Asx was 1:1 molar ratio in low protein concentration, and 2:2 molar ratio in high protein concentration (21). Crystal structure work on the deubiquitinase Calypso, the Drosophila counterpart of BAP1, and its activating deubiquitinase adaptor (Deubad) protein partner ASX have provided a structural basis to interpret studies demonstrating that the ASXL1/2 Deubad domains bind tightly to BAP1, and thereby activate the PR-DUB complex by forming a composite binding site for ubiquitin (21). As in our study, Foglizzo et al (21) showed that mutations at the juncture between DUB, Deubad, and ubiquitin have a deleterious effect on the ability of the PR-DUB to interact with ubiquitin.…”

Section: Discussionmentioning

confidence: 99%

Kinetics characterization of ASXL1/2-mediated allosteric regulation of BAP1 deubiquitinase

Peng

Cassel

McCracken

et al. 2020

Preprint

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32BAP1 is a ubiquitin hydrolase whose deubiquitinase activity is mediated by polycomb group-like 33 protein ASXL2. Cancer-related mutations/deletions of BAP1 lead to loss-of-function either by directly 34 targeting the catalytic (UCH) or ULD domains of BAP1, the latter disrupts binding to ASXL2, an 35 obligate partner for BAP1 enzymatic activity. However, the biochemical and biophysical properties of the 36 domains involved in forming the enzymatically active complex are unknown. Here we investigate the 37 molecular dynamics, kinetics and stoichiometry of these interactions. We demonstrate that the BAP1 and 38 ASXL2 domain/proteins or protein complexes produced in either bacteria or baculovirus are structurally 39 and functionally active. The interaction between BAP1 and ASXL2 is direct, specific, and stable to in 40 vitro biochemical and biophysical manipulations as detected by isothermal titration calorimetry, GST 41 association, and optical biosensor assays. Association of the ASXL2-AB box greatly stimulates BAP1 42 deubiquitinase activity. A stable ternary complex can be formed comprised of the BAP1-UCH, BAP1-43 ULD, and ASXL2-AB domains. Binding of the BAP1-ULD domain to the ASXL2-AB box is rapid, with 44 fast association and slow dissociation rates. Stoichiometric analysis revealed that one molecule of the 45 ULD domain directly interacts with one molecule of the AB Box. Real-time kinetics analysis of ULD/AB 46 protein complex to the UCH domain of BAP1, based on SPR, indicated that formation of the ULD/AB 47 complex with the UCH domain is a single-step event with fast association and slow dissociation rates.48 These structural and dynamic parameters implicate the possibility for future small-molecule approaches to 49 reactivate latent wild-type UCH activity in BAP-mutant malignancies. 50 51 The abbreviations used are: UCH, ubiquitin C-terminal hydrolase; ULD, UCH37-like domain; PcG, 52 polycomb group (PcG); polycomb repressive deubiquitinase (PR-DUB); ASXH, Asx homology domain; 53 PHD, plant homeo domain; PRC2, polycomb repressive complex 2; NLS, nuclear localization signals; 54 Bac, bacteria; Bv, baculovirus; SPR, surface plasmon resonance; ITC, isothermal titration calorimetry; 55 CD, circular dichroism; DLS, dynamic light scattering. 56 4 57 Introduction 58BAP1 was discovered as an ubiquitin hydrolase that associates with the RING finger domain of 59 BRCA1 and enhances BRCA1-mediated inhibition of breast cancer cell growth (1). The N-terminus of 60 BAP1 consists of a UCH domain (ubiquitin C-terminal hydrolase) that cleaves ubiquitin from ubiquitin-61 conjugated small substrates. BAP1 contains two protein-binding motifs for BARD1 and BRCA1, which 62 form a tumor suppressor heterodimeric complex (2), and a binding site for HCF1, which interacts with a 63 histone-modifying complex during cell division (3). The C-terminus of BAP1 contains two nuclear 64 localization signals and ULD (UCH37-like domain). The ULD domain interacts with ASXL family 65 members to form the polycomb group (PcG)-repressive deubiquiti...

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“…Association of the ULD and DEUBAD domains increases the affinity of the PR-DUB for ubiquitin and is required for deubiquitination [ 6 , 9 , 10 ]. At the C-terminus of their ULD domains, Calypso and BAP1 each have positively charged C-terminal extensions, which are required for binding the net negatively charged nucleosome [ 8 , 9 ]. A major difference in domain architecture between BAP1 and Calypso is an insertion of around 380 amino acids into the ULD domain of BAP1, which is proposed to enable a broader range of interactions by the mammalian protein [ 11 , 12 , 13 ] ( Figure 1 ).…”

Section: Pr-dub the Polycomb-repressive Deubiquitinasementioning

confidence: 99%

“…Two near-identical structures of Calypso bound to the DEUBAD of Asx were recently solved and provide structural insight into deubiquitination by the PR-DUB [ 8 , 23 ]. In these structures, the UCH domain of Calypso is linked to the ULD through a coiled-coil hairpin.…”

Section: Regulation Of Pr-dub Catalytic Activitymentioning

confidence: 99%

Molecular Regulation of the Polycomb Repressive-Deubiquitinase

Reddington

Fellner

Burgess

et al. 2020

IJMS

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Post-translational modification of histone proteins plays a major role in histone–DNA packaging and ultimately gene expression. Attachment of ubiquitin to the C-terminal tail of histone H2A (H2AK119Ub in mammals) is particularly relevant to the repression of gene transcription, and is removed by the Polycomb Repressive-Deubiquitinase (PR-DUB) complex. Here, we outline recent advances in the understanding of PR-DUB regulation, which have come through structural studies of the Drosophila melanogaster PR-DUB, biochemical investigation of the human PR-DUB, and functional studies of proteins that associate with the PR-DUB. In humans, mutations in components of the PR-DUB frequently give rise to malignant mesothelioma, melanomas, and renal cell carcinoma, and increase disease risk from carcinogens. Diverse mechanisms may underlie disruption of the PR-DUB across this spectrum of disease. Comparing and contrasting the PR-DUB in mammals and Drosophila reiterates the importance of H2AK119Ub through evolution, provides clues as to how the PR-DUB is dysregulated in disease, and may enable new treatment approaches in cancers where the PR-DUB is disrupted.

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A bidentate Polycomb Repressive-Deubiquitinase complex is required for efficient activity on nucleosomes

Cited by 32 publications

References 69 publications

Structural Basis for the Activation of the Deubiquitinase Calypso by the Polycomb Protein ASX

Structural Basis for the Activation of the Deubiquitinase Calypso by the Polycomb Protein ASX

Kinetics characterization of ASXL1/2-mediated allosteric regulation of BAP1 deubiquitinase

Molecular Regulation of the Polycomb Repressive-Deubiquitinase

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