Evolution-based Protein Design: Reverse Engineering Transthyretin for Hyperuricemia Therapeutic Applications

Transthyretins (TTRs) are vertebrate-exclusive proteins whose primary function is binding and transporting thyroid hormones (T3 and T4). They are widely studied due to their medical relevance concerning transthyretin amyloidosis, which affects several organs. Furthermore, TTRs stand as subjects of interest in evolutionary biology. Their origin traces back to the 5-hydroxyisourate hydrolase (HIUase), a ubiquitous enzyme responsible for the hydrolysis of 5-hydroxyisourate (5-HIU), a byproduct of the purine catabolic pathway. Approximately 500 million years ago, an ancestor HIUase gene underwent a duplication event, with one of the copies accumulating mutations and eventually giving rise to the TTR gene. As a result of their common ancestry, both proteins share many structural and biophysical similarities, as their monomeric subunit comprises eight beta-strands connected by seven loops, and one alpha-helix, while being found in vivo as homotetramers. Tetramer formation is stabilized by hydrophobic interactions between the pair of dimers, creating an internally charged cavity conducive to catalysis. In Homo sapiens, mutations in the uricase gene decreased the evolutionary pressure on other genes within the pathway, including HIUase. The lack of expression of these enzymes is associated with negative outcomes of uric acid accumulation such as gout and hyperuricemia. Current treatments are known to have a low coverage and efficiency of controlling uric acid pathogenesis, with even some patients being prone to high antigenicity. Taking this into account and leveraging the evolutionary parallels between TTRs and HIUases, we engineered two TTR mutants with HIUase function, while maintaining structural and sequence similarity to the wild-type H. sapiens TTR. By employing bioinformatics, phylogenetics, and experimental approaches we converted residues linked to thyroxine binding from TTR into catalytic residues from HIUase. The new enzymes were modeled in silico, and mutant sequences were cloned into pET28a-TEV, expressed in E. coli heterologous system, purified, and biophysically characterized through mass-spectrometry, circular dichroism and compared to an active wild-type HIUase through enzymatic assays. The designed enzymes would serve as biopharmaceuticals that could be administered alongside hyperuricemia treatments in order to mitigate the consequences of uric acid buildup and reduce potential adverse side effects common in current therapies.