Application of a Highly Selective Cathepsin S Two-step Activity-Based Probe in Multicolor Bio-Orthogonal Correlative...

Area of Science:

• Biochemistry
• Cell Biology
• Immunology

Background:

• Cathepsin S is a lysosomal cysteine protease crucial for immune cell function.
• Its roles in extracellular matrix breakdown and MHC II maturation require precise activity mapping.
• Existing methods struggle to delineate cathepsin S activity with high spatial resolution.

Purpose of the Study:

• To develop a potent and highly selective activity-based probe for cathepsin S.
• To apply this probe in multicolor bio-orthogonal correlative light-electron microscopy.
• To investigate the ultrastructural localization of cathepsin S activity in immune cells.

Main Methods:

• Design of a two-step activity-based probe based on LHVS, incorporating azide handles for click chemistry.
• Optimization of azide group positioning (P2 and P1) to enhance selectivity and detectability.
• Application of the optimized probe in multicolor bio-orthogonal confocal and correlative light-electron microscopy.

Main Results:

• A novel, selective activity-based probe for cathepsin S was successfully developed.
• Probe efficacy was significantly influenced by the positioning of click chemistry handles.
• The probe enabled ultrastructural localization of cathepsin S activity in bone marrow-derived dendritic cells.

Conclusions:

• The developed probe offers unprecedented spatial precision for studying cathepsin S activity.
• This tool facilitates the characterization of diverse cathepsin S functions in biological processes.
• The findings highlight the importance of probe design in activity-based protein profiling.