Targeted Protein Relocalization via Protein Transport Coupling

Subcellular protein localization regulates protein function and can be corrupted in cancers and neurodegenerative diseases. The localization of numerous proteins has been annotated, and pharmacologically relevant approaches for precise rewiring of localization to address disease-driving phenotypes would be an attractive targeted therapeutic approach. Molecules which harness the trafficking of a shuttle protein to control the subcellular localization of a target protein could provide an avenue for targeted protein relocalization for interactome-rewiring therapeutics. To realize this concept, we deploy a quantitative approach to identify features which govern the ability to hijack protein trafficking, develop a collection of shuttle proteins and ligands, and demonstrate relocalization of proteins bearing endogenous localization signals. Using a custom imaging analysis pipeline, we show that endogenous localization signals can be overcome through molecular coupling of target proteins to shuttle proteins containing sufficiently strong native localization sequences expressed in the necessary abundance. We develop nuclear hormone receptors as viable shuttles which can be harnessed with Targeted Relocalization Activating Molecules (TRAMs) to redistribute disease-driving mutant proteins such as SMARCB1Q318X, TDP43, and FUSR495X. Small molecule-mediated relocalization of FUSR495X to the nucleus from the cytoplasm reduced the number of cellular stress granules in a model of cellular stress. Using Cas9-mediated knock-in tagging, we demonstrate nuclear enrichment of both low abundance (FOXO3a) and high abundance (FKBP12) endogenous proteins via molecular coupling to nuclear hormone receptor trafficking. Finally, small molecule-mediated redistribution of NMNAT1 from nuclei to axons in primary neurons was able to slow axonal degeneration and pharmacologically mimic the WldS gain-of-function phenotype from mice resistant to certain types of neurodegeneration8. The concept of targeted protein relocalization could therefore nucleate approaches for treating disease through interactome rewiring.