All bacteria possess multiple ATP-dependent proteases that degrade cytosolic proteins. These enzymes help maintain protein homeostasis and regulate discrete pathways, including the expression of virulence phenotypes in pathogenic bacteria, and have emerged as attractive antibacterial targets. ATP-dependent proteases are able to selectively recognize substrate proteins and ignore non-substrate proteins, minimizing harmful or wasteful off-target proteolysis. Many substrates are recognized directly by short, unstructured terminal sequences, termed degrons. While a small number of degrons have been identified, there is little known about the overarching rules that allow proteases to effectively discriminate between valid degrons and the millions of other possible terminal sequences. To address this gap in our knowledge, we have developed a cell-based screening platform that will allow us to interrogate global degron specificity and define the sequence-based rules that govern recognition of protein substrates by ATP-dependent proteases. Our method incorporates a novel selection-based screen, in which a library of protein toxin bearing a randomized terminal tag is expressed in host bacteria. Accumulation of toxin in host cells causes cell death. However, toxins bearing bona fide degrons are proteolyzed by endogenous proteases, allowing cell survival. Bacteria expressing valid degron sequences are enriched over time, and identified by Next-Generation Sequencing. Here we describe the efficacy of our method in E. coli, supported by preliminary NGS data from pilot screening experiments. The information gathered from this method we ultimately help us understand the roles that ATP-dependent proteases play in individual pathogenic bacteria.

High-throughput discovery of sequences that promote proteolysis in bacteria

ACS Spring Meeting 2021

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