Reactive oxygen species (ROS) are transient, highly reactive intermediates or byproducts produced during oxygen metabolism. However, when innate mechanisms are unable to cope with sequestration of surplus ROS, it causes oxidative stress, where excess ROS damage biomolecules. Oxidized phosphatidylserine (PS), a pro-apoptotic “eat me” signal, is produced in response to elevated ROS, yet, little is known of its chemical composition and metabolism. Here, we report a small molecule that generates ROS in different mammalian cells, using which we detect, characterize and study oxidized PS in mammalian cells. We describe a chemical genetic screen to identify enzymes that regulate oxidized PS in mammalian cells, and find that the lipase ABHD12 hydrolyzes oxidized PS. We validate these findings in different physiological settings including primary peritoneal macrophages, and brains from Abhd12–/– knockout mice under inflammatory stress, and in the process functionally annotate an enzyme capable of regulating oxidized PS in vivo.

Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is a rare genetic human neurological disorder caused by null mutations to the Abhd12 gene, which encodes the integral membrane serine hydrolase enzyme ABHD12. Although the role that ABHD12 plays in PHARC is understood, the thorough biochemical characterization of ABHD12 is lacking. Here, we report the facile synthesis of mono-1-(fatty)acyl-glycerol lipids of varying chain lengths and unsaturation and use this lipid substrate library to biochemically characterize recombinant mammalian ABHD12. The substrate profiling study for ABHD12 suggested that this enzyme requires glycosylation for optimal activity and that it has a strong preference for very-long-chain lipid substrates. We further validated this substrate profile against brain membrane lysates generated from WT and ABHD12 knockout mice. Finally, using cellular organelle fractionation and immunofluorescence assays, we show that mammalian ABHD12 is enriched on the endoplasmic reticulum membrane, where most of the very-long-chain fatty acids are biosynthesized in cells. Taken together, our findings provide a biochemical explanation for why very-long-chain lipids (such as lysophosphatidylserine lipids) accumulate in the brains of ABHD12 knockout mice, which is a murine model of PHARC.

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The serine hydrolase (SH) superfamily is perhaps, one of the largest functional enzyme classes in all forms of life, and consists of proteases, peptidases, lipases, and carboxylesterases as representative members. Consistent with the name of this superfamily, all members, without any exception to date, use a nucleophilic serine residue in the enzyme active site to perform hydrolytictype reactions via a two-step ping-pong mechanism involving a covalent enzyme intermediate. Given the highly conserved catalytic mechanism, this superfamily has served as a classical prototype in the development of several platforms of the chemical proteomics technique, activitybased protein profiling (ABPP), to globally interrogate the functions of its different members in various native, yet complex, biological settings. While ABPP-based proteome-wide activity atlas' for SH activities are available in numerous organisms, including humans, to the best of our knowledge, such an analysis for this superfamily is lacking in any insect model. To address this, here, we initially report a bioinformatics analysis towards the identification and categorization of non-redundant SHs in Drosophila melanogaster. Following up on this in silico analysis, leveraging discovery chemoproteomics, we identify and globally map the full complement of SH activities during various developmental stages and in different adult tissues of Drosophila. Finally, as proof of concept of the utility of this activity atlas, we highlight sexual dimorphism in SH activities across different tissues in adult Drosophila melanogaster, and together, we prospect new research directions, resources and tools that this study can provide to the fly community.