Activity-based profiling of lipases in living cells

Area of Science:

• Biochemistry
• Proteomics
• Chemical Biology

Background:

• The ultimate goal of proteomics is to understand protein function in physiologically relevant contexts.
• Activity-based proteomics is a key strategy for achieving this by detecting enzyme activity directly within the proteome.
• Existing methods face challenges in probe properties affecting reactivity and cell permeability.

Purpose of the Study:

• To develop novel activity-based probes for detecting enzyme activity in intact living cells.
• To design probes with reporter tagging capabilities after target binding.
• To enable the characterization of lipolytic activities within the cellular environment.

Main Methods:

• Design and synthesis of activity-based probes with specific recognition, reactive, and reporter sites.
• Development of probes that mimic natural substrates, are small, and hydrophobic for cell membrane penetration.
• Establishment of a methodology for labeling and detecting target enzymes in intact living cells.

Main Results:

• Probes were engineered with reporter tagging functionalities post-enzyme binding.
• The developed probes exhibit properties suitable for labeling within living cells, including small size and hydrophobicity.
• A complete methodology for detecting lipolytic activities in intact living cells was successfully established.

Conclusions:

• Activity-based proteomics can be advanced using probes designed for post-binding reporter tagging.
• The developed methodology allows for the detection of enzyme activities, specifically lipolytic activities, in intact living cells.
• This approach offers a more physiologically relevant way to study protein function.