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S-glycosylation-based cysteine profiling reveals regulation of glycolysis by itaconate

Abstract

Itaconate has been recently recognized as an anti-inflammatory metabolite involved in the pathogen–macrophage interface. Due to its weak electrophilicity, itaconate could modify cysteines of the protein KEAP1 and glutathione, which contribute to its anti-inflammatory effect. However, the substrates of itaconate modification in macrophages have not been systematically profiled, which largely impedes the understanding of its roles in immune responses. Here, we developed a specific thiol-reactive probe, 1-OH-Az, for quantitative chemoproteomic profiling of cysteine modifications by itaconate, and provided a global portrait of its proteome reactivity. We found that itaconate covalently modifies key glycolytic enzymes and impairs glycolytic flux mainly through inhibition of fructose-bisphosphate aldolase A (ALDOA). Moreover, itaconate attenuates the inflammatory response in stimulated macrophages by impairing the glycolysis. Our study provides a valuable resource of protein targets of itaconate in macrophages and establishes a negative-feedback link between glycolysis and itaconate, elucidating new functional insights for this anti-inflammatory metabolite.

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Fig. 1: Development of 1-OH-Az as an efficient and unique probe for cysteinome profiling based on its S-glycosylation reactivity.
Fig. 2: Chemoproteomic profiling of itaconate-modified cysteines by competitive isoTOP-ABPP using 1-OH-Az.
Fig. 3: Itaconate modifies and inhibits ALDOA.
Fig. 4: Itaconate impairs glycolysis through modifying ALDOA.
Fig. 5: Itaconate’s anti-inflammatory effect is mediated by impairing glycolysis through ALDOA inhibition.

Data availability

The proteomics data (ID: 206029, 206030, 206031) has been deposited at https://chorusproject.org/pages/index.html. The data that support the findings of this study are available from the corresponding authors upon request.

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Acknowledgements

We thank the Computing Platform of the Center for Life Science for supporting the proteomics data analysis and Metabolomics Facility Center in National Protein Science Technology Center of Tsinghua University for C13 isotope tracing experiments. This work is supported by the National Key Research and Development Projects (grant no. 2016YFA0501500) to C.W and X.C., and (no. 2018YFA0507600) to X.C., and the National Natural Science Foundation of China (nos. 21521003, 81490740 and 21778004) to C.W. and (nos. 21425204, 21521003 and 21672013) to X.C., and a ‘1,000 Talents Plan’ Young Investigator Award to C.W.

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Authors and Affiliations

Authors

Contributions

W.Q., X.C. and C.W. conceived the project. W.Q. conducted most of the experiments unless specified otherwise. K.Q. carried out the LC–MS/MS analysis. Y.Z. carried out the gene cloning. W.J. cultured BMDMs under the guidance of Y-L.W. Y.C. performed IA-alkyne-based rdTOP-ABPP. B.C. synthesized 1-OH-Az and its derivatives. L.P. helped evaluate the 1-OH-Az labeling. N.C. synthesized UK-alkyne probe. Y.L. analyzed ALDOA structure and W.Z. performed ETD-based LC–MS/MS analysis. W.Q., X.C. and C.W. analyzed the data and wrote the manuscript with input from all the authors.

Corresponding authors

Correspondence to Xing Chen or Chu Wang.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–14

Reporting Summary

Dataset 1

Identification of the cysteines modified by 1-OH-Az and Ac4ManNAz in MCF-7 cells.

Dataset 2

Identification of the proteins modified by 1-OH-Az and Ac4ManNAz in MCF-7 cells.

Dataset 3

Competitive isoTOP-ABPP profiling of itaconate-modified cysteines using the 1-OH-Az probe in Raw264.7 cells with 250 μM of itaconate treatment.

Dataset 4

Competitive isoTOP-ABPP profiling of itaconate-modified cysteines using the 1-OH-Az probe in Raw264.7 cells with 1 mM of itaconate treatment.

Dataset 5

Competitive rdTOP-ABPP profiling of itaconate-modified cysteines using the IA-alkyne probe in Raw264.7 cells.

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Qin, W., Qin, K., Zhang, Y. et al. S-glycosylation-based cysteine profiling reveals regulation of glycolysis by itaconate. Nat Chem Biol 15, 983–991 (2019). https://doi.org/10.1038/s41589-019-0323-5

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