A Protein Capsid-Based Delivery System For Carrying Selenocysteine Into Cells

Small-molecule diselenides are redox-active compounds that can provoke a wide range of responses in cells.For example,selenocystamine and selenoglutathione have been shown to enhance oxidative protein folding in bacteria and yeast.In contrast,selenocystine,the diselenide dimer of selenocysteine（Sec）,is highly toxic to both bacteria and yeast,as well as a broad range of mammalian cells,with IC₅₀ values typically in the low micromolar range.Indeed,its cytotoxic activity makes selenocystine attractive as a potential anti-cancer drug.The amino acid selenocysteine（Sec）has received a lot of attention as a potential anti-cancer drug.The mechanism of selenocystine-induced cytotoxicity remains unclear.Misincorporation of Sec into proteins and metabolism of Sec by a dedicatedβ-lyase enzyme may contribute.Further,it has been shown that selenocystine can disrupt redox homeostasis,leading to overoxidation and apoptosis in the cell.The oxidative activity of selenocystine stems from the chemistry of the diselenide bond.Compared with disulfide bonds,diselenides are more thermodynamically stable,but also more kinetically reactive.Its broad cytotoxicity limits its usefulness as a therapeutic medicine.Therefore,Sec is an attractive candidate for drug delivery,but targeted delivery vehicles for Sec are lacking.Nanoparticles have been used to be a drug delivery system for clinical application.These nanoparticles provide a shell that envelops the drug.Protein capsids can form a nanoscale container for storage and delivery of many molecular cargoes.Their hollow supramolecular structures and high degree of symmetry provide a favorable condition for genetic and chemical modification.Conjugation of a drug to the inside of a protein capsid can protect the drug from the in vivo environment.Compared to synthetic polymers,protein capsids have the advantages of good biocompatibility,easy synthesis,and efficient elimination from the body.In addition,proteins are monodisperse.Protein capsids represent a promising class of nanoscale container for the storage and delivery of molecular cargoes.Their hollow supramolecular structures and high degree of symmetry provide a scaffold that can be readily modified by genetic or chemical methods.The capsid formed by Aquifex aeolicus lumazine synthase（AaLS）is built from 60 identical monomers that assemble as a docamer-of-pentamers and is an attractive starting point for engineering new drug delivery systems.Here,we demonstrate that the capsid formed by Aquifex aeolicus lumazine synthase（AaLS）can act as a nanocarrier for Sec.A previously reported variant of AaLS（AaLS-IC）,which contains a single cysteine per subunit that projects into the capsid interior,was modified by reaction with selenocystine to generate a selenosulfide conjugate between the capsid and selenocysteine（AaLS-IC-Sec）.Each AaLS-IC-Sec capsid contains approximately 48 selenocysteines,and efficient loading requires placement of the reactive cysteine inside of the protein capsid.A protein with the cysteine exposed to the outside cannot form the unique protein-drug conjugate by thiol-diselenide exchange reaction which is a significant advantage for developing AaLS-IC as a drug delivery system.Atmospheric oxygen are able to oxyidize reduced selenocystine very quickly and efficiently which can also benefit to the loading process.Encapsulated Sec can be quantitatively released from the capsid by common reducing agents,such as GSH or DTT,with a half-time of³ h.This property should be useful for the triggered release of selenocysteine in cancer cells,which tend to have higher levels of glutathione,compared to healthy cells.For four of the six cell lines tested,cell viability measurements indicated that AaLS-IC-Sec can go into the cells and release Sec in a toxic form.Fluorescence microscopy experiments revealed an intriguing correspondence between the cytotoxicity of AaLS-IC-Sec and the intracellular trafficking of the AaLS capsid.Cells that do not take up the capsid are insensitive to AaLS-IC-Sec.Moderately and highly sensitive cells accumulate the AaLS capsid in the cytoplasm and nucleus,respectively.This observed phenomenon can help us further modify AaLS-IC with intracellular targeting properties.Taken together,this study provides a promising foundation for the development of novel systems for the delivery of Sec into cells.