Tokushima University develops new technology for readily manipulating formation and dissolution of protein condensates

A research group led by Professor Tomohide Saio and Assistant Professor Motonori Matsusaki of the Institute of Advanced Medical Sciences, Tokushima University, and Do Thanh Tuan of the Graduate School of Medicine, Tokushima University, has developed "OptoChaperone," a tool that can readily manipulate protein droplet formation and dissolution by light irradiation. By enabling "dissolution of droplets that have been formed," which was previously difficult, the tool opens up possibilities for more detailed analysis of the causal relationship between the retention/elimination of phase separation and pathological aggregation. The results were published in the online edition of the Journal of the American Chemical Society.

It is known that cells respond to environmental changes and form protein condensates (droplets) by a molecular assembly process called biological phase separation (liquid-liquid phase separation) to modulate various biological functions such as stress response and signal transduction.

When this droplet-regulating mechanism gets disrupted, the condensates remain undissolved and abnormal aggregation occurs, which is believed to be the cause of various neurodegenerative diseases including ALS and Alzheimer's disease.

Meanwhile, the processes undergone by the protein droplets after their formation to eventually express their functions, as well as how dysregulation of protein droplets induces abnormal aggregate formation and results in cytotoxicity, remain unknown. To elucidate these mechanisms, several optogenetic tools have been developed for artificially inducing protein droplet formation by light, which is advantageous in spatiotemporal control at the cellular level. However, since most of these tools were primarily designed to induce droplet formation, it was difficult to dissolve the droplets once they were formed.

In this study, the group focused on the TF (Trigger Factor) chaperone, one of the molecular chaperones that suppress the aggregation of defective proteins in actual cells. They developed OptoChaperone by modifying this TF with an azobenzene derivative that changes its steric structure depending on the wavelength of light, and fusing it with a lid protein.

Irradiation with visible light (450 nm) opens the lid and activates the chaperone (ON state), leading to dissolution of the target protein droplets. Ultraviolet (UV) light closes the lid and inactivates the chaperone function (OFF state), allowing droplet formation.

They tested the efficacy of OptoChaperone on FUS and TDP43 (proteins related to the pathogenesis of ALS) and HSF1 (a stress response factor). The test demonstrated that the formation and dissolution of droplets could be reversibly regulated by simply switching the type of light, without any chemical additives or genetic modification of the target proteins. In addition, intracellular experiments have shown that regulation of condensation by OptoChaperone also affects cell fate determination (survival) under heat stress.

Various neurodegenerative diseases, including ALS, Parkinson's disease and Alzheimer's disease, are known to be caused by excessive accumulation of conformationally abnormal protein aggregates in the body.

Meanwhile, these disease-associated proteins function by reversibly forming and dissolving phase-separation droplets, contributing to maintenance of homeostasis. OptoChaperone has potential applications in a variety of studies, such as elucidation of life systems and disease mechanisms. The technology potentially contributes to the advancement in life science and biomolecular engineering and is also expected to promote drug discovery for neurodegenerative diseases in the future.

Journal Information
Publication: Journal of the American Chemical Society
Title: OptoChaperone—A Biohybrid Tool for Regulating Protein Condensates in Cells and In Vitro
DOI: /10.1021/jacs.6c04074