Direct measurement of deubiquitinating enzyme activity in intact cells using a protease-resistant, cell-permeable, peptide-based reporter

• Published in: Biochemical engineering journal 2019-11, Vol.151, p.107320, Article 107320
• Main Authors: Safa, Nora, Pettigrew, Jacob H., Gauthier, Ted J., Melvin, Adam T.
• Format: Article
• Language: English
• Subjects: Cell penetrating peptides; Deubiquitinating enzymes (DUBs); Fluorometry; Peptide-based biosensors; Protease-resilient peptides; Single cell analysis
• ISSN: 1369-703X; 1873-295X
• DOI: 10.1016/j.bej.2019.107320

[Display omitted] •New approach to measure deubiquitinating enzyme (DUB) activity in intact cells.•Peptide-based reporter incorporates a β-hairpin sequence motif that is a CPP and protectide.•Developed a kinetic model to account for both peptide uptake and reaction kinetics.•Demonstrated similar reaction kinetics to commercially available peptides in cell lysates.•Observed time- and concentration-dependent increase in intracellular fluorescence due to DUBs. Deubiquitinating enzymes (DUBs) regulate the removal of the polyubiquitin chain from proteins targeted for degradation. Current approaches to quantify DUB activity are limited to test tube-based assays that incorporate enzymes or cell lysates, but not intact cells. The goal of this work was to develop a novel peptide-based biosensor of DUB activity that is cell permeable, protease-resilient, fluorescent, and specific to DUBs. The biosensor consists of an N-terminal β-hairpin motif that acts as both a ‘protectide’ to increase intracellular stability and a cell penetrating peptide (CPP) to facilitate the uptake into intact cells. The β-hairpin was conjugated to a C-terminal substrate consisting of the last four amino acids in ubiquitin (LRGG) to facilitate DUB mediated cleavage of a C-terminal fluorophore (AFC). The kinetics of the peptide reporter were characterized in cell lysates by dose response and inhibition enzymology studies. Inhibition studies with an established DUB inhibitor (PR-619) confirmed the specificity of both reporters to DUBs. Fluorometry and fluorescent microscopy experiments followed by mathematical modeling established the capability of the biosensor to measure DUB activity in intact cells while maintaining cellular integrity. The novel reporter introduced here is compatible with high-throughput single cell analysis platforms such as FACS and droplet microfluidics facilitating direct quantification of DUB activity in single intact cells with direct application in point-of-care cancer diagnostics and drug discovery.