Design of improved therapeutic proteins and novel protein-based nanomaterials

Abstract

Proteins are involved in a myriad of biological processes such as catalysis, transport, regulation, defense, and providing structure to the cell. Most proteins need to fold into a defined 3D structure to perform such functions. However, this energetically directed process can be kinetically disturbed, resulting in the formation of stable misfolded supramolecular assemblies, including different types of protein aggregates. Protein aggregation stems from the establishment of aberrant intermolecular interactions and has been associated with the onset of several degenerative diseases. In addition, this unwanted reaction limits the development of proteins of biotechnological interest, impacting the production, storage, and commercialization of protein-based drugs. In this regard, strategies aimed to diminish the impact of protein aggregation on therapeutic proteins are pivotal to ensure their correct development as safe and active drugs. In the present doctoral thesis, we demonstrate that a structure-based aggregation predictor, which considers protein stability, successfully assists the redesign of two structurally unrelated proteins, improving their solubility without compromising their active conformation. This approach might replace the expensive trial-and-error assays employed by the pharmaceutical industry, providing an economical alternative for accelerating the development of protein-based drugs. Proteins are widely understood as the building blocks of life and can act as self-assembling entities involved in the creation of different supramolecular structures. Hence, there is an increasing interest in using polypeptides to build up functional and biocompatible nanomaterials; among them, protein inclusion bodies (IBs) have emerged as an attractive architecture. Traditionally considered useless protein deposits formed by misfolded conformations, recent data converge to indicate that IBs act as reservoirs of stable and active protein. These submicrometric particles are produced efficiently and cost-effectively, and are usually sustained by an amyloid-like scaffold where the protein of interest is trapped. Nevertheless, this amyloid conformation necessarily impacts the activity of the protein of interest and can be potentially cytotoxic. To overcome these drawbacks, we aimed at generating novel and improved functional IBs based on the α-helical architecture characteristic of coiled-coils. Using a naturally encoded coiled-coil domain as the scaffolding entity, α-helix-rich and biocompatible functional IBs were obtained in a ready-to-use form. Interestingly, coiled-coil-based IBs present a higher specific activity than their amyloid-like counterparts, since their assembly is guided by native interactions that do not interfere with the folding of the functional moieties. We demonstrate that these coiled-coil-inspired protein nanoparticles can display fluorescent and antibody-capturing activities simultaneously, being biocompatible, stable, and targeting specific antigens when decorated with a single or a combination of antibodies; thus, emerging as a promising technology for biomedical applications. Overall, the work described in the present thesis attempts to provide useful strategies aimed at (I) redesigning therapeutic proteins with enhanced biophysical properties employing in silico tools and (II) developing versatile and tunable multifunctional protein-based nanomaterials sustained by α-helical interactions.