Abstract B169: Engineering phase separation of multivalent signaling proteins through the solubility of folded protein domains

Abstract

Similar to three-dimensional membraneless organelles, it has been demonstrated that T-cell LAT signaling clusters form through multi-valency driven phase separation. This phase separated cluster was found to promote Arp2/3-complex driven actin assembly in vitro and correlated with increased ERK phosphorylation in cells. However, in this initial study, perturbations used to study phase separation also affect interactions known to be important for T-cell signaling, convoluting the impact of phase separation on signaling with effects due to changes in canonical biochemical interactions. To understand more precisely the role of phase separation in T-cell signaling, I will alter the solubility of folded domains through mutations of surface charged residues to modulate their phase separation propensities, independent of their high-affinity ligand binding interactions. To demonstrate the validity of this approach, I have first applied it to a synthetic system, based on multivalent interactions between the small ubiquitin-like modifier (SUMO) and the SUMO interaction motif (SIM), which will enable us to provide design principles that can be used on T-cell receptor signaling proteins. I demonstrate that it is possible to specifically alter the solubility of folded domains without altering their canonical molecular interactions. The solubility of individual folded domains serves as a predictor of the concentration threshold of phase separation, and phase separation can either be enhanced or abolished with a single mutation. Principles gained from this work on SUMO/SIM will be applied to multivalent proteins involved T-cell receptor signaling, namely Grb2 and Nck1, and the relationship between phase separation and signaling will be firmly established with future implications for rational engineering of T-cells. Citation Format: Jonggul Kim, Jonathon Ditlev, Michael K. Rosen. Engineering phase separation of multivalent signaling proteins through the solubility of folded protein domains [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B169.