Lipolytic and esterolytic activity-based profiling of murine liver

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• The liver plays a critical role in lipid metabolism, buffering energy fluctuations by storing fatty acids as triacylglycerol and secreting them as very low-density lipoprotein.
• The specific lipolytic enzymes responsible for mobilizing internal lipid stores within the liver remain largely unidentified.

Purpose of the Study:

• To identify and functionally annotate lipolytic and esterolytic enzymes in mouse liver subproteomes.
• To develop and apply activity-based proteomics methods for comprehensive profiling of enzyme activities in complex biological mixtures.

Main Methods:

• Utilized activity-based proteomics with fluorescent and biotinylated chemical probes targeting active enzyme sites.
• Analyzed fluorescently labeled proteomes using 2-DE (two-dimensional electrophoresis).
• Isolated biotinylated proteins via avidin-affinity capture for subsequent analysis.

Main Results:

• Identified a total of 37 proteins, with 15 detected by both fluorescent and biotinylated probe methods.
• 14 proteins were exclusively identified by 2-DE, and 8 by avidin-affinity isolation.
• Classified 12 enzymes as potential lipases and/or cholesteryl esterases based on their reactivity with specific activity probes.

Conclusions:

• Activity-based proteomics is effective for discovering and discriminating lipolytic and esterolytic enzymes in liver subproteomes.
• This study provides a foundational catalog of potential lipases and esterases involved in hepatic lipid metabolism.
• The identified enzymes are critical targets for understanding and potentially modulating liver lipid homeostasis.