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A chemical–genetic screen identifies ABHD12 as an oxidized-phosphatidylserine lipase

Nature Chemical Biology volume 15, pages 169–178 (2019)Cite this article

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Abstract

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, oxidative stress results, in which excess ROS damage biomolecules. Oxidized phosphatidylserine (PS), a proapoptotic ‘eat me’ signal, is produced in response to elevated ROS, yet little is known regarding its chemical composition and metabolism. Here, we report a small molecule that generates ROS in different mammalian cells. We used this molecule to detect, characterize and study oxidized PS in mammalian cells. We developed a chemical–genetic screen to identify enzymes that regulate oxidized PS in mammalian cells and found that the lipase ABHD12 hydrolyzes oxidized PS. We validated these findings in different physiological settings including primary peritoneal macrophages and brains from Abhd12–/– mice under inflammatory stress, and in the process, we functionally annotated an enzyme regulating oxidized PS in vivo.

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Fig. 1: Characterization of a ROS-generating probe (MGR1), and an inactive control probe (MGR2).
Fig. 2: Characterization and quantification of oxidized PS in mammalian cells after MGR1 treatment.
Fig. 3: Chemical–genetic screen to identify oxidized-PS lipases in mammalian cells.
Fig. 4: Biological validation of human ABHD12 as an oxidized-PS lipase.
Fig. 5: ABHD12 functions as an oxidized-PS lipase in vivo.

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Data availability

The authors declare that all the data that support the findings of this study are available in the paper, associated supplementary information files and datasets.

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Acknowledgements

This work was supported by grants from the Wellcome Trust DBT India Alliance (IA/I/15/2/502058 to S.S.K.), DST-SERB (ECR/2016/001261 to S.S.K.; EMR/2015/000668 to H.C.), DBT (BT/PR15848/MED/29/1025/2016 to H.C.) and DST-FIST (Infrastructure Development to IISER Pune Biology Department). B. F. Cravatt (The Scripps Research Institute) is thanked for providing chemical compounds, inhibitors and ABHD12-knockout mice used in this study, and for insightful comments on the manuscript. N. Balasubramanian (IISER Pune) is thanked for access to the EVOS Imaging System for the cellular fluorescence experiments. The National Facility for Gene Function in Health and Disease, IISER Pune, is thanked for maintaining and providing mice for this study. G.R., A.K.S. and A.R. acknowledge research fellowships from the Council for Scientific and Industrial Research (CSIR), Government of India, and N.M. acknowledges a research fellowship from DBT, Government of India.

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Author notes

  1. These authors contributed equally: Dhanashree S. Kelkar, Govindan Ravikumar, Neelay Mehendale, Shubham Singh.

Authors and Affiliations

  1. Department of Biology, Indian Institute of Science Education and Research (IISER), Pune, India

    Dhanashree S. Kelkar, Neelay Mehendale, Shubham Singh, Alaumy Joshi, Amol Mhetre, Abinaya Rajendran & Siddhesh S. Kamat

  2. Department of Chemistry, Indian Institute of Science Education and Research (IISER), Pune, India

    Govindan Ravikumar, Ajay Kumar Sharma & Harinath Chakrapani

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Contributions

D.S.K., N.M., S.S., A.J., A.R. and S.S.K. performed the biochemical experiments and analyzed the data. G.R., A.K.S., A.M. and H.C. synthesized and chemically characterized all the chemical compounds in this study. D.S.K. and S.S.K. performed and analyzed the proteomics data. S.S.K. and H.C. conceived the project and designed the experiments. S.S.K. wrote the paper, to which all authors provided input.

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Correspondence to Siddhesh S. Kamat.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–26

Reporting Summary

Supplementary Note

Synthetic procedures

Supplementary Dataset 1

LC-MS/MS based chemoproteomic characterization

Supplementary Dataset 2

MRM transitions and quantitation of PS, lyso-PS and oxidized PS lipids

Supplementary Dataset 3

Highly focused library of lipase inhibitors tested in the chemical genetic screen

Supplementary Dataset 4

Complete proteomics data sets from RAW264.7 cells

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Kelkar, D.S., Ravikumar, G., Mehendale, N. et al. A chemical–genetic screen identifies ABHD12 as an oxidized-phosphatidylserine lipase. Nat Chem Biol 15, 169–178 (2019). https://doi.org/10.1038/s41589-018-0195-0

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