Protease Activity Profiling Via Programmable Phage Display

Abstract

Endopeptidases catalyze the internal cleavage of proteins, playing pivotal roles in protein turnover, substrate maturation and the activation of signaling cascades. A broad range of biological functions in health and disease are controlled by proteases, yet assays to characterize their activities at proteomic scale do not yet exist. To address this unmet need, we have developed SEPARATE (Sensing EndoPeptidase Activity via Release and recapture using flAnking Tag Epitopes), which uses monovalent phage display of the entire human proteome at 90-aa peptide resolution. We demonstrate that SEPARATE is compatible with several human proteases from distinct catalytic classes, including Caspase-1, ADAM17, and Thrombin. Both well-characterized and newly identified substrates of these enzymes were detected in the assay. SEPARATE was used to discover a non-canonical Caspase-1 substrate, the E3 ubiquitin ligase HUWE1, a key mediator of apoptotic cell death. SEPARATE is a novel methodology to enable efficient, unbiased assessment of endopeptidase activity using a phage-displayed proteome.