Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling

  • Cell Chem Biol. 2017 Feb 16;24(2):231-242. doi: 10.1016/j.chembiol.2017.01.002.
Rhushikesh A Kulkarni  1 ,  Andrew J Worth  2 ,  Thomas T Zengeya  1 ,  Jonathan H Shrimp  1 ,  Julie M Garlick  1 ,  Allison M Roberts  1 ,  David C Montgomery  1 ,  Carole Sourbier  3 ,  Benjamin K Gibbs  3 ,  Clementina Mesaros  2 ,  Yien Che Tsai  4 ,  Sudipto Das  5 ,  King C Chan  5 ,  Ming Zhou  5 ,  Thorkell Andresson  5 ,  Allan M Weissman  4 ,  W Marston Linehan  3 ,  Ian A Blair  2 ,  Nathaniel W Snyder  6 ,  Jordan L Meier  7
Affiliations
  • 1. Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
  • 2. Penn SRP Center, Center for Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia, PA 19104, USA.
  • 3. Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20817, USA.
  • 4. Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
  • 5. Protein Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
  • 6. Drexel University, A.J. Drexel Autism Institute, 3020 Market Street, Philadelphia, PA 19104, USA.
  • 7. Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA. Electronic address: [email protected].
PMID: 28163016 DOI: 10.1016/j.chembiol.2017.01.002
Abstract

Non-enzymatic protein modification driven by thioester reactivity is thought to play a major role in the establishment of cellular lysine acylation. However, the specific protein targets of this process are largely unknown. Here we report an experimental strategy to investigate non-enzymatic acylation in cells. Specifically, we develop a chemoproteomic method that separates thioester reactivity from enzymatic utilization, allowing selective enrichment of non-enzymatic acylation targets. Applying this method to Cancer cell lines identifies numerous candidate targets of non-enzymatic acylation, including several Enzymes in lower glycolysis. Functional studies highlight malonyl-CoA as a reactive thioester metabolite that can modify and inhibit glycolytic enzyme activity. Finally, we show that synthetic thioesters can be used as novel reagents to probe non-enzymatic acylation in living cells. Our studies provide new insights into the targets and drivers of non-enzymatic acylation, and demonstrate the utility of reactivity-based methods to experimentally investigate this phenomenon in biology and disease.

Keywords
Warburg effect; acetylation; acylation; epigenetics; glycolysis; malonylation; metabolism; non-enzymatic; reactivity-based protein profiling; thioester.
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