A FRET sensor for the real-time detection of long chain acyl-CoAs and synthetic ABHD5 ligands

Summary

Intracellular long-chain acyl-coenzyme As (LC-acyl-CoAs) are thought to be under tight spatial and temporal controls, yet the ability to image LC-acyl-CoAs in live cells is lacking. Here, we developed a fluorescence resonance energy transfer (FRET) sensor for LC-acyl-CoAs based on the allosterically regulated interaction between α/β hydrolase domain-containing 5 (ABHD5) and Perilipin 5. The genetically encoded sensor rapidly detects intracellular LC-acyl-CoAs generated from exogenous and endogenous fatty acids (FAs), as well as synthetic ABHD5 ligands. Stimulation of lipolysis in brown adipocytes elevated intracellular LC-acyl-CoAs in a cyclic fashion, which was eliminated by inhibiting PNPLA2 (ATGL), the major triglyceride lipase. Interestingly, inhibition of LC-acyl-CoA transport into mitochondria elevated intracellular LC-acyl-CoAs and dampened their cycling. Together, these observations reveal an intimate feedback control between LC-acyl-CoA generation from lipolysis and utilization in mitochondria. We anticipate that this sensor will be an important tool to dissect intracellular LC-acyl-CoA dynamics as well to discover novel synthetic ABHD5 ligands.

Graphical abstract

Highlights

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  A FRET sensor that detects long-chain acyl-CoAs (LC-CoAs) and synthetic ABHD5 ligands

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  The sensor reports ligand-dependent interaction between ABHD5 and PLIN5

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  Activating lipolysis in brown adipocytes (BAs) raises LC-CoAs in a cyclical fashion

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  LC-CoAs provide feedback regulation from mitochondria to lipid droplets in BAs

Motivation

Intracellular long-chain acyl-coenzyme As (LC-acyl-CoAs) are thought to be under tight spatial and temporal controls, yet the ability to image LC-acyl-CoAs in live cells is lacking. We sought to create a fluorescence resonance energy transfer (FRET) sensor of LC-acyl-CoAs and synthetic ABHD5 ligands based on the reversible interactions between ABHD5 and PLIN5. The sensor allows dynamic imaging of intracellular LC-acyl-CoA levels in targeted subcellular compartments. In addition, since ABHD5 has emerged as a therapeutic target for metabolic disease and cancer, a robust FRET-based sensor could aid in the identification of novel synthetic and natural ABHD5 ligands.

Abstract

Mottillo et al. develop a single-molecule FRET sensor based on the ligand-dependent interaction between ABHD5 and PLIN5. The sensor reports intracellular long-chain acyl-CoA (LC-CoA) levels and detects synthetic ABHD5 ligands. Real-time imaging in brown adipocytes reveals feedback inhibition between LC-CoA generation from lipolysis and uptake in mitochondria.