Humanized archaeal ferritin as a tool for cell targeted delivery

Abstract

Human ferritins have been extensively studied to be used as nanocarriers for diverse applications and could represent a convenient alternative for targeted delivery of anticancer drugs and imaging agents. However, the most relevant limitation to their applications is the need for highly acidic experimental conditions during the initial steps of particle/cargo assembly, a process that could affect both drug stability and the complete reassembly of the ferritin cage. To overcome this issue the unique assembly of Archaeoglobus fulgidus ferritin was genetically engineered by changing a surface exposed loop of 12 amino acids connecting B and C helices to mimic the sequence of the analogous human H-chain ferritin loop. This new chimeric protein was shown to maintain the unique, cation linked, association-dissociation properties of Archaeoglobus fulgidus ferritin occurring at neutral pH values, while exhibiting the typical human H-homopolymer recognition by the transferrin receptor TfR1. The chimeric protein was confirmed to be actively and specifically internalized by HeLa cells, thus representing a unique nanotechnological tool for cell-targeted delivery of possible payloads for diagnostic or therapeutic purposes. Moreover, it was demonstrated that the 12 amino acids' loop is necessary and sufficient for binding to the transferrin receptor. The three-dimensional structure of the humanized Archaeoglobus ferritin has been obtained both as crystals by X-ray diffraction and in solution by cryo-EM.

Excimer based fluorescent pyrene–ferritin conjugate for protein oligomerization studies and imaging in living cells

“…11,12,30,31 At the two-fold axis of the dimer, in the middle of the B helix, a single point conservative mutation (M54C) was introduced for site-selective N-(1-pyrenyl)maleimide (NPM) labeling. The mutated residues were positioned far from any inter-subunit contacts or loop regions in order to avoid any interference with the cage assembly, at a 14 A distance between two b-carbons of two C54, enough for a p-stacking interaction between two pyrene molecules (Fig.…”

“…12 The mutated ferritin from Pyrococcus furiosus PfFtP77C and Archaeoglobus fulgidus AfFtM54C, already available in our lab, were puried as previously reported. …”

Enzyme Encapsulation by a Ferritin Cage

“…Recently, it was shown that Tfr1‐mediated internalization can be transferred to non‐human ferritins as well. Grafting of only 12 amino acids of an external subunit‐spanning loop from HsFtn onto the ferritin of the archaeon Archaeoglobus fulgidus (AfFtn; Figure ) enabled Tfr1 binding and cytosolic internalization …”

Molecular Entrapment in Thermophilic Ferritin for Nanoformulation in Photodynamic Therapy

“…Human ferritin owing to the presence of transferrin receptor (TfR1) binding domain is known to be internalized by transferrin receptor mediated endocytosis . It has also been shown that unengineered AfFtn does not follow transferrin receptor mediated endocytosis and incorporation of transferrin receptor binding domain is required for it to be endocytosed by the human transferrin receptor mediated endocytosis pathway . The uptake mechanism of AfFtn although is found to be receptor mediated in this study, further studies to identify the exact endocytosis path are required which would also help in designing the cages for specific receptor targeting.…”