Vlachos, E. N. | Search bioRxiv

bioRxiv2026

Functional diversification of two Lon homologs enhances stress adaptation in Pseudomonas aeruginosa

The Lon protease is a highly conserved ATP-dependent protease that contributes to protein quality control and regulatory processes across all domains of life. The opportunistic pathogen Pseudomonas aeruginosa, along with other members of the Pseudomonadales, encodes AsrA (aminoglycoside-induced stress response ATP-dependent protease), a second LonA-type protease in addition to canonical Lon. Although AsrA is upregulated by aminoglycoside stress, its biochemical activity, substrate spectrum, and cellular functions have remained elusive. Here, we demonstrate that AsrA is a temperature- and antimicrobial stress-induced protease with specialized functions and partial redundancy with Lon. Quantitative proteomics revealed a distinct AsrA substrate profile, while comparative biochemical analyses showed that AsrA and Lon share a substantial number of substrates in vitro but differ in their degradation kinetics, indicating divergent substrate preferences. Among the proteins preferentially degraded by AsrA is the quorum-sensing anti-activator QslA, and we show that AsrA-dependent QslA degradation under tobramycin stress induces quorum-sensing gene expression. Together, our findings demonstrate how duplication of a conserved protease can generate specialized regulatory functions through differential expression and substrate preference, expanding the proteolytic network that enables bacterial adaptation to stress.

microbiology↗

bioRxiv2024

Design of linear and cyclic peptide binders of different lengths only from a protein target sequence

Structure prediction technology has revolutionised the field of protein design, but key questions such as how to design new functions remain. Many proteins exert their functions through interactions with other proteins, and a significant challenge is designing these interactions effectively. While most efforts have focused on larger, more stable proteins, shorter peptides offer advantages such as lower manufacturing costs, reduced steric hindrance, and the ability to traverse cell membranes when cyclized. However, less structural data is available for peptides and their flexibility makes them harder to design. Here, we present a method to design both novel linear and cyclic peptide binders of varying lengths based solely on a protein target sequence. Our approach does not specify a binding site or the length of the binder, making the procedure completely blind. We demonstrate that linear and cyclic peptide binders of different lengths can be designed with nM affinity in a single shot, and adversarial designs can be avoided through orthogonal in silico evaluation, tripling the success rate. Our protocol, EvoBind2 is freely available https://github.com/patrickbryant1/EvoBind.

bioinformatics↗