A Cell-Permeable β-Hairpin Peptide Biosensor for Real-Time, Single-Cell Quantification of Proteasome Activity

Measuring proteasome activity in intact, live cells provides essential information to gain insight into protein degradation dynamics, cellular stress response, and drug response. This study presents a modular, peptide-based biosensor that integrates a β-hairpin cell-penetrating peptide (CPP), a proteasome-specific recognition sequence, a Rhodamine 110 fluorophore, and a CPP-enhancer sequence to improve cellular uptake, which allows for sensitive and selective quantification of proteasome activity in intact cells. Two different peptides were synthesized that differed in the CPP-enhancer sequence and were characterized in two model cell lines known to exhibit enhanced proteasome activity: OPM.2 (multiple myeloma) and A549 (lung cancer). Both peptide-based biosensors were found to effectively quantify proteasome activity in cell lysates and live, intact cells using fluorometry and fluorescence microscopy. A comparison of biosensor performance was performed using metrics such as maximum attainable signal, background noise levels, and signal to noise ratio (S/N) that demonstrated that the peptide biosensor with the CPP enhancing sequence with fewer positively charged amino acid residues was the superior design. The unique incorporation of a CPP-enhancing sequence not only enabled efficient cellular entry, but it also influenced the observed fluorescence signal, underscoring the importance of selecting biosensor sequence elements to achieve accurate proteasome quantification in complex biological systems. The proposed biosensor offers compatibility with multiple detection platforms that position it as a versatile tool for studying proteasome activity in complex biological systems.