Artificial organelles based on hybrid protein nanoparticles

Compartmentalization is one of the main strategies by which nature allows for control over many biological processes. As such, organelles are essential for the proper functioning of living cells. Researchers have been inspired by nature to construct artificial organelles to either add new functionalities or to correct dysfunctional processes in living cells. This can be done by either using synthetic components to produce artificial organelles in vitro or in situ by using components that can be produced within a cell. The latter approach requires the biosynthesis of the artificial organelle compartments. As such, protein-based nanocages are excellent candidates for engineering towards this end. Chapter 1 highlights the various approaches that have been taken to develop a variety of protein compartments into in vivo artificial organelles.
An interesting candidate for development into a protein-based artificial organelle system is an engineered variant of the cowpea chlorotic mottle virus (CCMV) that is fused to a stimulus-responsive and stabilizing elastin-like polypeptide (ELP) domain. In chapter 2 the potential of three previously engineered hybrid ELP-CCMV variants for the development into in vivo artificial organelles is evaluated by studying the self-assembly behavior and stability of capsids in physiologically relevant conditions. We aimed at finding a protein nanocage that can self-assemble under physiological conditions and is sufficiently stable to remain assembled for extended periods. UV-vis spectrometric studies, dynamic light scattering analysis, and transmission electron microscopy measurements demonstrated that two hybrid variants self-assemble into stable capsids at pH 7.5, physiological NaCl concentration, and 37 °C. The most hydrophobic variant also remained stable in a cell culture medium. As such, this VW1-VW8 ELP-CCMV variant was found to be most suitable for development into artificial organelles.
Chapter 3 highlights the most important findings of a study of the self-assembly dynamics of capsids of the VW1-VW8 ELP-CCMV variant during pH-induced size changes. Distinguishing between the larger pH-induced T = 3 capsids and the smaller ELP-induced T = 1 capsids without affecting the stability of the T = 1 capsids during analysis was not trivial. Therefore, we first developed a protocol for experimentally evaluating the capsid dynamics during size increase and decrease with size exclusion chromatography (SEC). The optimized protocol was used to study the capsid size over prolonged periods to evaluate at what time scale(s) capsids underwent a size change. We observed that, although the capsids did not completely disassemble, a size shift was possible, with capsids increasing in size upon a pH decrease and a size reduction induced by pH increase over two months. Finally, our experimental results were described with a mathematical model based on the classical nucleation theory (CNT) of virus capsids to apprehend the possible underlying mechanisms via which the VW1-VW8 ELP-CCMV capsids change in size dynamically. Combined, these results indicate that the capsids are still dynamic and can interact with their environment, which is promising for the development of responsive or adaptive artificial organelle systems.
We developed an efficient cargo-encapsulation strategy to enable loading our VW1-VW8 ELP-CCMV capsids with active cargoes, which is described in chapter 4. Although cargo loading methodologies for CCMV capsids already existed, most of the strategies are either dependent on the disassembly into dimers or not feasible in vivo. Therefore, the previously developed approaches were not compatible with either our VW1-VW8 ELP-CCMV system or with in vivo artificial organelle systems. This required the exploration of alternative cargo-loading strategies. We demonstrated that the cargo protein enhanced green fluorescent protein (mEGFP) can be incorporated into VW1-VW8 ELP-CCMV capsids in vitro either by Sortase A-mediated functionalization of the coat proteins in capsids or by mixing the empty capsid with an mEGFP-VW1-VW8 ELP-CCMV fusion protein. Both of these methods are based on the self-assembly dynamics that were described in chapter 3. Furthermore, an in vivo cargo encapsulation method was developed based on the co-expression and co-assembly of the VW1-VW8 ELP-CCMV protein and mEGFP-fused coat proteins. Especially the latter cargo incorporation strategy is promising for in vivo artificial organelle development.
The co-expression approach was expanded to incorporate an enzyme, PylD, into the VW1-VW8 ELP-CCMV capsids, thereby creating active nanoreactors. In chapter 5 our approach towards coupling VW1-VW8 ELP-CCMV nanoreactor activity to the activation of an intracellular response is discussed. The incorporated PylD enzyme catalyzes the final step in the biosynthesis of pyrrolysine, a noncanonical amino acid (ncAA). We incorporated this enzyme into the VW1-VW8 ELP-CCMV capsids and evaluated the activity of the resulting nanoreactors for the production of an ncAA in vitro. The activity of the encapsulated PylD enzyme was enhanced compared to nonencapsulated PylD variants. The ncAA that was produced by the nanoreactors was employed to induce EGFP expression in cell lines expressing an orthogonal pyrrolysine tRNA/synthetase pair and an EGFP with a genetically encoded amber stop codon that could be overwritten by the incorporation of the ncAA. Our VW1-VW8 ELP-CCMV-based artificial organelle precursor thus has the potential to induce an intracellular response.
Finally, the great advantage of a protein-based artificial organelle system compared to (partially) synthetic systems, is the potential of genetic incorporation of intracellular functionalities. Chapter 6 explores the protection of a destabilized intracellular cargo protein as an example of an intracellular functionality of our VW1-VW8 ELP-CCMV capsids. N-terminal degradation domains were introduced to destabilize the previously used mEGFP-VW1-VW8 ELP-CCMV fusion protein in E. coli, resulting in rapid degradation after the protein is expressed. We attempted to protect the cargo protein against degradation by co-expressing it with VW1-VW8 ELP-CCMV capsids, which would lead to their co-assembly. The same approach was employed in a mammalian cell line, HEK293T. Although interesting initial results were obtained, several aspects in both the E. coli and HEK293T system still require attention to realize a successful system wherein VW1-VW8 ELP-CCMV-based artificial organelles fulfill an intracellular function.
Together, the different research chapters demonstrate the progress that has been made towards the development of ELP-CCMV particles into functional in vivo artificial organelles. In chapter 7, the remaining challenges are identified and the significance of the results to the research field is reflected. Finally, future directions for protein-based artificial organelle engineering are explored.