Sahrawat, A. S. | bioRxiv Science

bioRxiv2026

Triacylglycerol fatty acid composition modulates ATGL regioselectivity and diacylglycerol output

Adipose triglyceride lipase (ATGL) catalyses the first and rate-limiting step of intracellular triacylglycerol (TAG) hydrolysis, yielding diacylglycerol (DAG) and a free fatty acid (FA). The site of TAG cleavage determines the regioisomeric identity of the DAG product, with distinct consequences for downstream signalling and lipid metabolism. Here, we show that ATGL prefers specific fatty acyl chain lengths and that this preference contributes to its regioselectivity on heterogeneous TAG substrates. Structural modelling identified a substrate-binding pocket comprising a hydrophilic anchor and a hydrophobic tunnel whose dimensions favour long-chain FAs. Mass spectrometry-based analysis of DAG products generated by ATGL from defined TAG substrates revealed an intrinsic preference for terminal (sn-1/sn-3) ester bond cleavage. Acyl-chain compatibility modulated this preference, with poorly accommodated terminal chains increasing the relative contribution of sn-2 cleavage. Consequently, ATGL generated predominantly sn-2-retaining DAG species from heterogeneous TAG substrates containing long-chain fatty acyl chains, including substantial amounts of the sn-1,2-DAG regioisomer, which has not previously been associated with ATGL activity. These findings link the fatty acyl chain composition of TAGs to the DAG regioisomer profile generated by ATGL-mediated lipolysis and provide substrate-dependent parameters for more species-resolved models of lipid metabolism.

biochemistry↗

bioRxiv2026

Acid phospholipase A1 promotes lysosomal membrane catabolism

The molecular mechanisms of lysosomal glycerophospholipid (GPL) catabolism are incompletely understood. Here, we report that acid phospholipase A1 (APLA1), formerly known as palmitoyl-protein thioesterase 2 (PPT2), is required for efficient GPL degradation. Deletion of APLA1 in human cells results in excess accumulation of phospholipids within lysosomes # a pathological condition termed phospholipidosis. APLA1 activity depends on interactions with negatively charged GPLs and is inhibited by phospholipidosis-inducing cationic amphiphilic drugs. Hydrolysis of zwitterionic, but not anionic, GPLs requires co-activation of APLA1 by the lysosome-specific lipid bis(monoacylglycero)phosphate. Upon pharmacological mTORC inhibition, which increases lysosomal GPL turnover, APLA1-deficient cells exhibit massive accumulation of multilamellar membranes in lysosomes and reduced cytosolic triacylglycerol stores. APLA1 acts in concert with lysosomal phospholipase A2 (PLA2G15). Combined APLA1/PLA2G15-deficiency leads to a severe reduction in acid phospholipase A1/A2 activity, thereby exacerbating phospholipidosis. Our observations provide detailed mechanistic insights into lysosomal GPL catabolism, a crucial pathway for maintaining lipid homeostasis.

biochemistry↗