RESUMEN
Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilizers.
Asunto(s)
Proteínas 14-3-3 , Proteínas Proto-Oncogénicas c-mdm2 , Péptidos/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismoRESUMEN
Protein post-translational modifications (PTMs) play an intricate role in a diverse range of cellular processes creating a complex PTM code that governs cell homeostasis. Understanding the molecular build-up and the critical factors regulating this PTM code is essential for targeted therapeutic design whereby PTM mis-regulation is prevalent. Here, we focus on Pin1, a peptidyl-prolyl cis-trans isomerase whose regulatory function is altered by a diverse range of PTMs. Through employing advanced mass spectrometry techniques in combination with fluorescence polarization and enzyme activity assays, we elucidate the impact of combinatorial phosphorylation on Pin1 function. Moreover, two phosphorylation sites were identified whereby Ser71 phosphorylation preceded Ser16 phosphorylation, leading to the deactivation of Pin1's prolyl isomerase activity before affecting substrate binding. Together, these findings shed light on the regulatory mechanisms underlying Pin1 function and emphasize the importance of understanding PTM landscapes in health and disease.
Asunto(s)
Peptidilprolil Isomerasa de Interacción con NIMA , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Peptidilprolil Isomerasa de Interacción con NIMA/química , Fosforilación , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
Molecular glues are powerful tools for the control of protein-protein interactions. Yet, the mechanisms underlying multi-component protein complex formation remain poorly understood. Native mass spectrometry (MS) detects multiple protein species simultaneously, providing an entry to elucidate these mechanisms. Here, for the first time, covalent molecular glue stabilization was kinetically investigated by combining native MS with biophysical and structural techniques. This approach elucidated the stoichiometry of a multi-component protein-ligand complex, the assembly order, and the contributions of covalent versus non-covalent binding events that govern molecular glue activity. Aldehyde-based molecular glue activity is initially regulated by cooperative non-covalent binding, followed by slow covalent ligation, further enhancing stabilization. This study provides a framework to investigate the mechanisms of covalent small molecule ligation and informs (covalent) molecular glue development.