A platform for post-translational spatiotemporal control of cellular proteins

Abstract

Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for target-specific control of protein destination. This platform is based on bifunctional molecules comprising a target-specific nanobody and universal sequences determining target subcellular localization or degradation rate. We demonstrate that nanobody-mediated localization depends on the expression level of the target and the nanobody, and the extent of target subcellular localization can be regulated by combining multiple target-specific nanobodies with distinct localization or degradation sequences. We also show that this platform for nanobody-mediated target localization and degradation can be regulated transcriptionally and integrated within orthogonal genetic circuits to achieve the desired temporal control over spatial regulation of target proteins. The platform reported in this study provides an innovative tool to control protein subcellular localization which will be useful to investigate protein function and regulate large synthetic gene circuits.

Targeted Protein Relocalization via Protein Transport Coupling

“…In the context of controlling subcellular localization, correcting diseased phenotypes directly resulting from protein mislocalization or imparting beneficial function through protein relocation could expand the range of therapeutic options. A selection of tools for studying protein sequestration upon different stimuli with synthetic protein fusions to nuclear hormone receptor ligand binding domains [30][31][32] , LOV2 domains [33][34][35] , or binding sites for localization sequence containing nanobodies 36 , have enabled fundamental studies of altered protein localization. Early work employing chemically-induced proximity between FRB and FKBP12 fused to arrayed localization sequences driven by rapamycin demonstrated the potential of small molecule control over localization programming [37][38][39] .…”