[P006] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P03-420] Versatile spatiotemporal control of protein functions via sterically bulky caging for medical applications

○Kosuke Habuchi¹, Chiharu Moriyama², Ryotaro Yamamoto^1,2, Akimitsu Okamoto², Satoshi Yamaguchi¹ (1. The University of Osaka (Japan), 2. The University of Tokyo (Japan))

Keywords:

Proteins,Photoactivation,Mammalian cells,Caging

[Purpose] Spatiotemporal control of protein function is a powerful tool for a wide range of medical applications, including protein drug delivery systems and regenerative medicine. In particular, photoresponsive protein activation techniques are promising for the selective use of protein functions at desired times and locations, for example, in the formation of complexartificial organoids, as well as in site-specific gene editing and vaccination. Among these approaches, “caging”—the reversible inactivation of proteins using photolabile protecting groups—has emerged as a useful strategy. However, conventional protein caging methods face significant challenges because small protecting groups must be precisely introduced into the active site. This typically requires complex genetic engineering techniques, such as the incorporation of unnatural amino acids. These approaches are often cumbersome, limited to proteins with known structures, and frequently result in reduced activity after irradiation due to residual mutations. Therefore, a simple and versatile method for protein caging is needed for practical applications.

[Method] To overcome the limitations of conventional protein caging methods, we developed a sterically bulky caging approach using a biotinylated photodegradable protecting reagent (biotinylated caging reagent, BCR) [1]. In this method, the target protein is modified with BCR via a photocleavable linker to introduce a biotin moiety. The biotinylated protein is then mixed with streptavidin, which binds strongly to biotin and sterically covers the protein surface, resulting in effective inactivation of the target protein. Thus, proteins can be simply andefficiently caged without the need for genetic engineering. Upon light irradiation, the photocleavable linker is cleaved, and both the biotinylated protecting group and streptavidin are released, leading to the restoration of protein activity. This approach enables versatile and structure-independent protein caging for diverse biomedical applications.

[Results] We have successfully applied this sterically bulky caging technology to a variety ofproteins, achieving reliable photoresponsiveness in all cases. For transferrin (Tf), an iron
transport protein, we controlled intracellular uptake via the transferrin receptor, allowing Tf and its loaded cargo to accumulate inside cells only after light irradiation[2]. In addition, we applied this method to saporin (Sap), a ribosome-inactivating protein; when caged Sap was introduced into cells, apoptosis was selectively induced upon light exposure [3]. These results demonstrate successful photoactivation of protein functions both inside and outside
cells. In this poster presentation, we will present recent progress in applying this approach to other proteins, including gene-editing enzymes.

References: [1] S. Takamori, et al., Chem. Comm., 49, 3013 (2013); [2] S. Yamaguchi, et al.,
Bioconj. Chem., 32, 1535 (2021); [3] S. Yamaguchi, et al., ChemBioChem, 23, 476 (2022).