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

Area of Science:

• Biochemistry
• Cell Biology
• Biotechnology

Background:

• Proteasome activity is crucial for protein degradation, cellular stress response, and drug efficacy.
• Accurate measurement of proteasome activity in intact cells is vital for biological research.

Purpose of the Study:

• To develop and characterize a modular, peptide-based biosensor for sensitive and selective quantification of proteasome activity in intact cells.
• To compare the performance of two biosensor designs differing in their cell-penetrating peptide (CPP) enhancer sequences.

Main Methods:

• Synthesis of two peptide-based biosensors incorporating a CPP, proteasome recognition sequence, and Rhodamine 110 fluorophore.
• Characterization of biosensor performance in OPM.2 (multiple myeloma) and A549 (lung cancer) cell lines using fluorometry and fluorescence microscopy.
• Evaluation of biosensor performance based on signal-to-noise ratio, maximum signal, and background noise.

Main Results:

• Both peptide biosensors successfully quantified proteasome activity in cell lysates and live cells.
• The biosensor with a CPP-enhancing sequence containing fewer positively charged amino acid residues demonstrated superior performance.
• The CPP-enhancing sequence significantly influenced cellular uptake and fluorescence signal.

Conclusions:

• The developed peptide biosensor is a versatile tool for studying proteasome activity in complex biological systems.
• Optimizing CPP-enhancer sequences is critical for achieving accurate and sensitive proteasome quantification.
• The biosensor is compatible with multiple detection platforms for broad applicability.