S-acylation of ATGL at Cys15: mechanism of activation and its relationship to ABHD5 coactivation

Abstract

Adipose triglyceride lipase (ATGL) catalyzes the first and rate-limiting step of triacylglycerol hydrolysis and is therefore a central regulator of lipid droplet catabolism in hepatocytes. Recent work identified S-acylation of ATGL at Cys15 as essential for lipase activity and lipid droplet homeostasis, but the mechanism by which this modification supports ATGL function remained unresolved. In particular, it was unknown whether Cys15 S-acylation influences ATGL activation through its major coactivator, α/β-hydrolase domain-containing protein 5 (ABHD5), and whether the requirement at Cys15 reflects a chemically specific need for cysteine modification or a more general requirement for local hydrophobicity. This thesis investigated the role of Cys15 S-acylation in ATGL activation and its relationship to ABHD5 coactivation. First, the effect of the S-acylation-deficient mutant C15S on ATGL interaction with and coactivation by ABHD5 was examined using confocal microscopy, co-immunoprecipitation, fluorescence lifetime imaging microscopy–Förster resonance energy transfer (FLIM-FRET), and cell-free lipase assays. Second, hydrophobic substitutions at Cys15 (C15F, C15L, and C15W) were tested to determine whether local hydrophobicity could partially substitute for S-acylation. Third, a preliminary microscopybased screen of candidate acyl-protein thioesterases was performed to identify potential negative regulators of ATGL deacylation. C15S retained localization to lipid droplets and co-immunoprecipitated with ABHD5, indicating that loss of S-acylation did not abolish association between the two proteins. However, FLIM-FRET showed that C15S did not exhibit the same degree of close-range proximity to ABHD5 observed for wild-type ATGL. Wild-type ATGL showed robust ABHD5-dependent activation, whereas C15S exhibited only weak stimulation, indicating that Cys15 S-acylation is required for full catalytic activation. Hydrophobic substitutions preserved lipid droplet localization and reduced lipid droplet retention relative to C15S, consistent with partial functional rescue. C15F mutant particularly restored FRETdetectable proximity to ABHD5 and partially recovered lipase activity, supporting the idea that hydrophobicity at position 15 contributes to ATGL activation, although it does not fully replace the effect of S-acylation. Finally, the thioesterase screen did not identify an obvious candidate regulator under the conditions tested. Together, these findings support a model in which Cys15 S-acylation promotes ATGL activation not by controlling lipid droplet targeting, but by enabling a productive functional relationship with ABHD5 and by contributing local hydrophobic character near the Nterminal catalytic region. This work refines the mechanistic understanding of ATGL regulation in hepatocytes and provides new insight into how defective ATGL activation may contribute to hepatic lipid accumulation and steatotic liver disease.