RESUMO
Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering, and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. By a modular combination of a tandem fusion of two oligomeric proteins, which forms phase-separated synthetic protein condensates in cells, with a chemically induced dimerization tool, we first created a chemogenetic protein condensate system that can rapidly recruit target proteins from the cytoplasm to the condensates by addition of a small-molecule dimerizer. We next coupled the protein-recruiting condensate system with an engineered proximity-dependent protease, which gave a second protein condensate system wherein target proteins previously expressed inside the condensates are released into the cytoplasm by small-molecule-triggered protease recruitment. Furthermore, an optogenetic condensate system that allows reversible release and sequestration of protein activity in a repeatable manner using light was constructed successfully. These condensate systems were applicable to control protein activity and cellular processes such as membrane ruffling and ERK signaling in a time scale of minutes. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step toward tailor-made engineering of synthetic protein condensate-based soft materials with various functionalities for biological and biomedical applications.
Assuntos
Células Artificiais/química , Células Artificiais/metabolismo , Proteínas/química , Proteínas/metabolismo , Animais , Células COS , Engenharia Celular , Chlorocebus aethiops , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Células NIH 3T3 , Proteínas/genética , Frações Subcelulares/metabolismoRESUMO
The development of methods that allow detection of ligand-target engagement in cells is an important challenge in chemical biology and drug discovery. Here, we present a Golgi recruitment (G-REC) assay in which the ligand binding to the target protein can be visualized as Golgi-localized fluorescence signals. We show that the G-REC assay is applicable to the detection of various ligand-target interactions, ligand affinity comparison among distinct protein isoforms, and the monitoring of unmodified drug-target engagement in cells.
Assuntos
Complexo de Golgi/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Trimetoprima/química , Trimetoprima/metabolismo , Fluorescência , Complexo de Golgi/química , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/química , Células HeLa , Humanos , Ligantes , Microscopia de Fluorescência , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Trimetoprima/síntese químicaRESUMO
The syntheses of various types of hexakis(4-functionalized-phenyl)benzenes 1 and hexakis[4-(4'-functionalized-phenylethynyl)phenyl]benzenes 2 by the cobalt-catalyzed cyclotrimerization of diarylacetylenes and by the Sonogashira coupling reaction of 1e with arylacetylenes, respectively, are described. X-ray crystallographic analysis showed that host 1e or 2f forms a 2-D network by unique I...I or CH...O=C interactions, respectively.
