The tumor suppressor p53 is regulated by phosphorylation-dependent protein-protein interactions, including via binding to 14-3-3 adaptor proteins, which can tune p53 activity. Molecular glue (MG)-induced stabilization of 14-3-3/client interactions offers an attractive strategy to probe such networks, but cellular engagement is often constrained by context-dependent phosphorylation and interaction occupancy. Here, we engineered phosphorylation- and MG-dependent 14-3-3 interaction cassettes (IC1 and IC2) and fused them to p53 to promote recruitment of endogenous 14-3-3 proteins in human cells. Biochemical characterization establishes high-affinity binding of the phosphorylated cassettes to 14-3-3 and enhanced in vitro stabilization by the 14-3-3 molecular glue 3'-deacetylated fusicoccin-A (FC-A). In HEK293T cells, Flag-p53-IC1 and Flag-p53-IC2 co-immunoprecipitated native 14-3-3 proteins. Mutation of the cassette's phospho-accepting serine to alanine abolished binding, confirming phosphorylation dependent recruitment. Transcriptomic profiling of transiently transfected cells reveals cassette-dependent remodeling of a p53-associated gene expression landscape. Together, these results establish a modular, MG- and phosphorylation-dependent platform for engaging 14-3-3 in a p53 context and for evaluating how chemical stabilization translates from biochemical interaction control to cellular pathway-level readouts.
Diana C. Muñoz-Lasso, Yan Ni, Glenn Weber et al.· ACS Chemical Biology· 0 citations
Proteins lacking defined ligandable pockets remain challenging drug targets. Here, we develop a molecular glue-based PROTAC (MGPROTAC) approach that chemically conjugates a molecular glue stabilizer to a VHL-recruiting ligand to capture and ubiquitinate the 14-3-3/Estrogen receptor α (ERα) complex. Our designed MGPROTACs engage a composite interface between 14-3-3 and the disordered F-domain of ERα, promoting cooperative complex formation and targeted ubiquitination. Biophysical characterization revealed distinct linker-dependent cooperativities across the MGPROTAC series, which influenced both cellular permeability and ubiquitination efficiency. Cryo-EM of the most cooperative MGPROTAC uncovered de novo VHL–14-3-3ζ contacts, while molecular dynamics simulations rationalize the stabilizing interactions underlying cooperativity. Strikingly, fine-tuning linker design enables selective ubiquitination of distinct complex subunits. These findings establish a structural and mechanistic framework for integrating molecular glue and PROTAC principles, expanding the scope of drug discovery to previously intractable protein complexes. Proteins lacking defined ligandable pockets are challenging drug targets. Here the authors combine molecular glue-stabilizer and PROTAC strategies to enable ubiquitination of the 14-3-3/estrogen receptor α complex, revealing cooperative linker-dependent activity and a structural basis for targeted E3 ligase recruitment.
Carlo J. A. Verhoef, Charlotte Crowe, M. Nakasone et al.· Nature Communications· 0 citations
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