Organelle-specific activity-based protein profiling in living cells

Area of Science:

• Biochemistry
• Cell Biology
• Chemical Biology

Background:

• Lysosomes are key organelles involved in cellular degradation and disease.
• Measuring specific enzyme activity within lysosomes remains challenging.
• Activity-based probes offer a way to profile enzyme function in situ.

Purpose of the Study:

• To develop a multimodal activity-based probe for targeting acidic organelles.
• To measure subcellular native enzymatic activity using fluorescence microscopy and mass spectrometry.
• To investigate cathepsin activity during autophagy.

Main Methods:

• Synthesis of a cathepsin-reactive probe with a clickable alkyne.
• Localization studies using structured illumination microscopy (SIM) in J774 cells.
• Quantitative analysis of probe labeling and cathepsin activity via mass spectrometry.
• Monitoring changes in enzyme activity during starvation-induced autophagy.

Main Results:

• The probe selectively accumulated in lysosomes of J774 mouse macrophage cells.
• Mass spectrometry confirmed high selectivity for cathepsins B and Z.
• Starvation-induced autophagy led to increased cathepsin activity and protein labeling.
• The probe enabled real-time monitoring of enzymatic activity in a disease-relevant pathway.

Conclusions:

• The developed activity-based probe is effective for measuring lysosomal cathepsin activity.
• This approach holds significant potential for studying diseases linked to lysosomal dysfunction.
• Multimodal probes offer a powerful strategy for in situ enzymatic profiling in organelles.