Article Highlight | 24-Jun-2025
Measurement of ATGL activity using adiposomes
image: A Protein expression of wild-type ATGL and mutants induced by IPTG. Wild-type ATGL, S47D, S87A and S87D (a), S47A and S87A (b), S430D and SMT3 (c), S430A and S430D (d) were expressed in bacteria cells. B Bacterial lysates of wild-type ATGL and mutants used for the TAG hydrolase assay. The content of ATGL and mutants was standardized by scanning the density of protein bands.
Credit: Xuejing Ma，Zelun Zhi，Shuyan Zhang，Pingsheng Liu
Key Findings:
- Improved ATGL activity measurement: Traditional methods utilizing lipid emulsions, micelles, or purified lipid droplets exhibit limitations such as heterogeneous lipid interfaces, difficulty in manipulating lipid composition, and interference from protein interactions. In contrast, the adiposome platform closely mimics the structure of native lipid droplets, allowing for precise control of lipid composition and providing a more accurate representation of cellular lipid and energy homeostasis.
- Introduction of SMT3 tag: To address the aggregation and precipitation of ATGL fusion proteins, an SMT3 tag was incorporated at the N-terminus. This modification enhances the solubility of ATGL and circumvents the loss of activity associated with affinity chromatography purification.
- Utilization of bacterial lysates: Due to the relatively low activity of purified ATGL, bacterial lysates were employed as a source of ATGL. The content of ATGL in these lysates was quantified through densitometric scanning of stained gels.
- Validation of the adiposome platform: The superiority of the adiposome platform for measuring ATGL activity was demonstrated through comparison with other methods, including lipid emulsions, micelles, and purified lipid droplets. The adiposome platform exhibited lower radioactivity, higher accuracy, and a structure more analogous to native lipid droplets.
- Analysis of ATGL mutants: Mutants of ATGL at reported phosphorylation sites were constructed and their activities were analyzed using the adiposome platform. The results indicated that mutations at S47 (S47A and S47D) and S87 (S87A) significantly decreased enzymatic activity, highlighting the critical role of these residues in regulating ATGL activity.
The work entitled “Measurement of ATGL activity using adiposomes”was published on Biophysics Reports (published in February, 2023).