Designing Protease-Triggered Protein Cages

Abstract

Proteins that self-assemble into enclosed polyhedral cages, both naturally and by design, are garnering attention for their prospective utility in the fields of medicine and biotechnology. Notably, their potential for encapsulation and surface display are attractive for experiments that require protection and targeted delivery of cargo. The ability to control their opening or disassembly would greatly advance the development of protein nanocages into widespread molecular tools. Toward the development of protein cages that disassemble in a systematic manner and in response to biologically relevant stimuli, here we demonstrate a modular protein cage system that is opened by highly sequence-specific proteases, based on sequence insertions at strategically chosen loop positions in the protein cage subunits. We probed the generality of the approach in the context of protein cages built using the two prevailing methods of construction: genetic fusion between oligomeric components and (non-covalent) computational interface design between oligomeric components. Our results suggest that the former type of cage may be more amenable than the latter for endowing proteolytically controlled disassembly. We show that a successfully designed cage system, based on oligomeric fusion, is modular with regard to its triggering protease. One version of the cage is targeted by an asparagine protease implicated in cancer and Alzheimer's disease, whereas the second version is responsive to the blood-clotting protease, thrombin. The approach demonstrated here should guide future efforts to develop therapeutic vectors to treat disease states where protease induction or misregulation occurs.

Programming interchangeable and reversible heterooligomeric protein self-assembly using a bifunctional ligand

“…Previous studies reported that a metal-chelator, reducing agent, or protease can trigger protein disassembly as external stimuli. [63][64][65] Similarly, we explored whether such changes could regulate the heteroassembly. The addition of 13 mM citrate (26% v/v) prior to or during dCA/KDGA assembly in TAPS buffer signicantly perturbed the PPI formation (Fig.…”

Self-assembling biomolecules for biosensor applications

“…Reprinted with permission from ref. [ 52 ]; Copyright 2022, American Chemical Society. c Schematic illustration of formation of ferritin cage-based activatable probes.…”

“…The Yeates group introduced protease-cleavable sequences into proteins designed to form protein cages through self-assembly, which allowed the nanostructures to disassemble responding to the specific enzymes (Fig. 3 b) [ 52 ]. Depending on the type of introduced sequence, the protein cages exhibited selective changes in the supramolecular structure under conditions associated with diseases, such as cancer, Alzheimer’s disease, and blood coagulation.…”

Recent advances in porous molecular cages for photocatalytic organic conversions

“…They exhibit favorable properties and possess promising potential for applications in the fields of supramolecular chemistry and porous materials. [20][21][22][23][24][25][26] The homogeneity of porous molecular cages enables their use in solution-based processing and mechanistic studies, where they can preorganize substrates, stabilize reactive intermediates, and increase local concentrations. As a result, these cages have found widespread application in enzyme-mimicking catalysis.…”