bioRxiv Science⌕ Search

Biology subjects

Owens, D. D. G.

Publications and source records attributed to Owens, D. D. G..

4 recordsLinked to original sources

Selective degradation of BRD9 by a DCAF16-recruiting targeted glue: mode of action elucidation and in vivo proof of concept

Prospective discovery of molecular glues degraders for a specific therapeutic target protein of interest is an emerging strategy in drug discovery. Modification of pre-existing ligands with fragments that can alter the protein surface can lead to the creation of novel compounds ( targeted glues) able to induce neo-interactions between the target and an E3 ligase, resulting in targeted protein degradation. By screening a library of potential BRD9 targeted glue compounds, we have discovered a potent and selective, reversibly covalent BRD9 degrader, AMPTX-1. Co-immunoprecipitation-mass spectrometry experiments demonstrated that cell treatment with AMPTX-1 induces selective recruitment of BRD9 to the E3 ligase DCAF16. Degradation is dependent on the engagement of the surface Cys58 of DCAF16 and formation of a covalent adduct to DCAF16 is facilitated by the ternary complex formation with BRD9. BRD9 degradation is achieved in vivo after oral dosing, demonstrating that covalent recruitment of DCAF16 is a viable strategy for targeted protein degradation and can be achieved with drug-like, orally bioavailable compounds with promising in vivo activity.

biochemistry↗

Interplay between β-propeller subunits WDR26 and muskelin regulates the CTLH E3 ligase supramolecular complex

The Pro/N-degron recognizing C-terminal to LisH (CTLH) complex is an E3 ligase of emerging interest in the developmental field and for targeted protein degradation (TPD) modalities. The human CTLH complex forms distinct supramolecular ring-shaped structures dependent on the multimerization of WDR26 or muskelin {beta}-propeller proteins. Here, we find that, in human cells, CTLH complex E3 ligase activity is dictated by a dynamic exchange between WDR26 and muskelin in tandem with muskelin autoregulation. Proteomic experiments revealed that complex-associated muskelin protein turnover is a major ubiquitin-mediated degradation event dependent on the CTLH complex in unstimulated HeLa cells. We observed that muskelin and WDR26 binding to the scaffold of the complex is interchangeable, indicative of the formation of separate WDR26 and muskelin complexes, which correlated with distinct proteomes in WDR26 and muskelin knockout cells. We found that mTOR inhibition-induced degradation of Pro/N-degron containing protein HMGCS1 is distinctly regulated by a muskelin-specific CTLH complex. Finally, we found that mTOR inhibition also activated muskelin degradation, likely as an autoregulatory feedback mechanism to regulate CTLH complex activity. Thus, rather than swapping substrate receptors, the CTLH E3 ligase complex controls substrate selectivity and its autoregulation through exchanging its {beta}-propeller oligomeric subunits WDR26 and muskelin.

molecular biology↗

A chemical probe to modulate human GID4 Pro/N-degron interactions

The CTLH complex is a multi-subunit ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via the substrate receptor GID4. Recently, focus has turned to this complex as a potential mediator of targeted protein degradation, but the role GID4-mediated substrate ubiquitylation and proteasomal degradation plays in humans has thus far remained unclear. Here, we report PFI-7, a potent, selective, and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation enabled the identification of dozens of endogenous GID4-interacting proteins that bind via the GID4 substrate binding pocket, only a subset of which possess canonical Pro/N-degron sequences. GID4 interactors are enriched for nuclear and nucleolar proteins including RNA helicases. GID4 antagonism by PFI-7 altered protein levels of several proteins including RNA helicases as measured by label-free quantitative proteomics, defining proteins that are regulated by GID4 and the CTLH complex in humans. Interactions with GID4 via Pro/N-degron pathway did not result in proteasomal degradation, demonstrating that CTLH interactors are regulated through a combination of degradative and non-degradative functions. The lack of degradation of GID4 interactors highlights potential challenges in utilizing GID4-recruiting bifunctional molecules for targeted protein degradation. Going forward, PFI-7 will be a valuable research tool for defining CTLH complex biology and honing targeted protein degradation strategies.

molecular biology↗

Dynamic Runx1 chromatin boundaries affect gene expression in hematopoietic development

The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increased in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells was associated with the formation of tissue-specific sub-TADs over Runx1, a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletions of promoter-proximal CTCF sites at the sub-TAD boundaries had no obvious effects on E-P interactions but led to partial loss of domain structure, mildly affected gene expression, and delayed hematopoietic development. Together, our analyses of gene regulation at a large multi-promoter developmental gene revealed that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.

developmental biology↗