Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling.
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.
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
Rhushikesh A
RA
Kulkarni
Penn SRP Center, Center for Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Andrew J
AJ
Worth
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
Thomas T
TT
Zengeya
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
Jonathan H
JH
Shrimp
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
Julie M
JM
Garlick
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
Allison M
AM
Roberts
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
David C
DC
Montgomery
Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20817, USA.
Carole
C
Sourbier
Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20817, USA.
Benjamin K
BK
Gibbs
Penn SRP Center, Center for Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Clementina
C
Mesaros
Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
Yien Che
YC
Tsai
Protein Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
Sudipto
S
Das
Protein Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
King C
KC
Chan
Protein Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
Ming
M
Zhou
Protein Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA.
Thorkell
T
Andresson
Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
Allan M
AM
Weissman
Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20817, USA.
W Marston
WM
Linehan
Penn SRP Center, Center for Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Ian A
IA
Blair
Drexel University, A.J. Drexel Autism Institute, 3020 Market Street, Philadelphia, PA 19104, USA.
Nathaniel W
NW
Snyder
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA. Electronic address: jordan.meier@nih.gov.
Jordan L
JL
Meier
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000215:Acylation; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D040901:Proteomics; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000214:Acyl Coenzyme A; D004952:Esters; D013438:Sulfhydryl Compounds
Warburg effect; acetylation; acylation; epigenetics; glycolysis; malonylation; metabolism; non-enzymatic; reactivity-based protein profiling; thioester