Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC–MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein–protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.
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The authors declare that all data supporting the findings of this study are available within the paper and its supplementary information files. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifiers PXD009378 (Supplementary Datasets 1–4) and PXD009202 (Supplementary Dataset 5). All of the data are accessible in the supplemental data sets (Supplementary Datasets 1–7) and can further be explored using our web-based resource (https://ccr2.cancer.gov/resources/Cbl/proteomics/fumarate).
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The authors thank C. Grose (Protein Expression Laboratory) for cloning and preparation of plasmid DNA, T. Archer (NIEHS) for the gift of the SMARCC1 and SNF5 plasmids, B. Weinberg (MIT) for the gift of the pLKO.1 puro plasmid (Addgene plasmid # 8453), A. Roberts, J. Garlick, and T. Zengeya (NCI) for assisting with preliminary studies. This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research (ZIA BC011488-05, ZIA BC011038-10) and the CCR FLEX Program. Support for E.W. was provided by the NIH (1R01GM117004 and 1R01GM118431-01A1).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figures 1–10
FH-regulated cysteines identified by comparative profiling of FH−/− HLRCC cell line (UOK262) and a FH+/+ rescue HLRCC cell line (UOK262WT); n = 3 independent experiments.
Compiled list of S-succinated cysteine residues previously characterized in the literature, and annotation with chemoproteomic data (if available).
Sequences used for motif analysis, as well as results for analyses of conservation based functional impact (FI), gene ontology (GO), and genomic lesions found in covalent fumarate targets in kidney cancer.
Fumarate-sensitive cysteines identified by competitive profiling of HEK-293 cells treated and untreated with fumarate (1 mM); n = 4 independent experiments.
Peptides identified as targets of S-succination in MudPIT LC–MS/MS analyses of HLRCC cell (UOK262 and UOK268) proteomes.
Analysis of transcripts co-regulated by FH and SNF5 in publicly accessible RNA-seq datasets.
Solvent-exposed surface area analysis of FH-regulated (Supplementary Dataset 1), exogenous fumarate sensitive (Supplementary Dataset 4), and hyperreactive cysteines.
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Kulkarni, R.A., Bak, D.W., Wei, D. et al. A chemoproteomic portrait of the oncometabolite fumarate. Nat Chem Biol 15, 391–400 (2019). https://doi.org/10.1038/s41589-018-0217-y
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