Lasso Peptide Engineering: Mutating Astexin-1 to Serve as a Caspase-3 Inhibitor

Abstract

Lasso peptides are a class of ribosomally synthesized natural products that are made by various bacteria. The stable knotted structure and myriad bioactivities make lasso peptides an interesting target of study. One potential therapeutic application is to engineer lasso peptides as protease inhibitors. The unique threaded construction of lasso peptides makes them resilient to proteolytic cleavage. Consequently, lasso peptides designed to bind to certain proteases might resist cleavage and prevent the enzymes from cutting other substrates. A protease of interest is caspase-3, which is implicated in many neurodegenerative disorders including Alzheimer’s disease. Caspase-3 has specificity for proteins with the four amino acid sequence, DEVD. This study attempted to integrate the DEVD segment into the constrained lasso peptide structure to inhibit caspase-3. The work focused on astexin-1, the largest lasso peptide to date. Unlike most lasso peptides, astexin-1 is very polar, making it a likely candidate to accept the polar caspase-3 binding sequence, DEVD. Additionally, single mutations were created in order to perform structure-activity relationship analysis on astexin-1. Exploring the amenability of astexin-1 to mutations is important in understanding its structure and the ability to redesign it as an engineered, stable scaffold. DEVD was incorporated into the atxA1 precursor gene and expressed in E. coli in the hopes of producing the astexin-1 peptide with the desired binding sequence. Mass spectrometry results showed no evidence of astexin-1 in either the supernatants or lysates of the mutant cells. However, many single mutations on both the ring and loop region of astexin-1 were tolerated. As astexin-1 is tolerable of amino acid substitution, it can be engineered for a variety of purposes. Furthermore, manipulation of single mutations and the flexible specificity of caspase-3 allowed for the insertion of recognition sites that deviated from the canonical sequence. These results indicate that astexin-1 is amenable to modification and a potential scaffold for various biological epitopes.