Engineering protein nanocages as carriers for biomedical applications

Bhaskar, Sathyamoorthy; Lim, Sierin

S Bhaskar and S Lim regulating the storage and release of iron to maintain homeostasis. These findings have shed light on the idea that proteins can be used to encapsulate and deliver active molecules in vivo. The first X-ray diffraction data of horse spleen ferritin were reported in 1943 12 and the structure in 1984, 13 but it was not until 1991 that the crystal structure of human ferritin (1FHA) was first reported and the cagelike architecture was clearly elucidated. 14 Since then, numerous ferritins and ferritin-like proteins have been developed for exogenous applications other than iron storage. 9 The globular, multisubunit caged ferritin stores iron in an insoluble non-toxic state while keeping it bioavailable within cells by readily converting it to its soluble form. The iron in the central cavity is maintained as small crystalline particles in an organic ferric oxyhydroxide form. Ferritin can withstand high temperature and a wide range of pH levels for limited periods without significant disruption to its caged quaternary structure, which is formed by 24 subunits. These subunits self-assemble into a hollow spherical structure with an outer diameter of 12 nm and an internal cavity with a diameter of 8 nm 5 ( Figure 2 ). Ferritin's iron core is approximately the size of its internal diameter. This critical diameter and several molecular characteristics, including electrophoretic mobility, solubility and antigenicity, are the most significant properties of the protein shell. 13 The self-assembly and disassembly properties of the ferritin nanocage can be controlled by metal ions, thereby aiding the uptake and release of active molecules from the protein structure. 17 Similarly, the structural and biochemical properties of ferritin protein are used for tailoring it to a wide range of applications, from the synthesis of nanoparticles to the design of vaccines in biomedicine. 18 E2 protein. The E2 protein is derived from the E2 core domain (dihydrolipoamide acetyltransferase) of the pyruvate dehydrogenase multienzyme complex. The protein is formed by 24 subunits in E. coli or 60 subunits in Geobacillus stearothermophilus that self-assemble into a hollow structure with a cubic core or an icosahedron with twelve 5-nm openings (PDB ID 1B5S), respectively. 19 Pyruvate dehydrogenase enzyme complex is composed of three enzymes: pyruvate decarboxylase (E1), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3) (Figure 3 ). It has been shown that the complex can be produced separately and the component enzymes can be reassembled in vitro to form the native structure. 19 Because E1 and E3 do not dissociate from each other in the presence of the enzyme E2, it is evident that the latter forms the structural core of the assembled complex. G. stearothermophilus is a thermophilic organism. Hence, the pyruvate dehydrogenase complex derived from it has innate stability that allows it to survive under extreme conditions. Unlike other nanocages such as viral capsids, the modification of the external surface with functional ligands does not hinder the assembly of the E2 core domain. This property is identical to that in its natural state, in which the E2 domain is linked to two distinct folding protein domains at its surface. 19 Similar to other natural protein nanocages, E2 protein nanocages can be subjected to genetic or chemical modifications to enable several functions such as drug loading and specific targeting protein attac