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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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.

Author information

Author notes

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

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.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Siddhesh S. Kamat.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–26

  2. Reporting Summary

  3. Supplementary Note

    Synthetic procedures

  4. Supplementary Dataset 1

    LC-MS/MS based chemoproteomic characterization

  5. Supplementary Dataset 2

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

  6. Supplementary Dataset 3

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

  7. Supplementary Dataset 4

    Complete proteomics data sets from RAW264.7 cells

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https://doi.org/10.1038/s41589-018-0195-0