Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles

Abstract

Cells produce electrophilic products with the potential to modify and affect the function of proteins. Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify the sites in the proteome that are most sensitive to electrophilic modification requires more quantitative methods. Here we describe a competitive activity–based profiling method for quantifying the reactivity of electrophilic compounds against >1,000 cysteines in parallel in the human proteome. Using this approach, we identified a select set of proteins that constitute 'hot spots' for modification by various lipid-derived electrophiles, including the oxidative stress product 4-hydroxy-2-nonenal (HNE). We show that one of these proteins, ZAK kinase, is labeled by HNE on a conserved, active site–proximal cysteine and that the resulting enzyme inhibition creates a negative feedback mechanism that can suppress the activation of JNK pathways normally induced by oxidative stress.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Competitive isoTOP-ABPP for quantitative mapping of cysteine–lipid-derived electrophile (LDE) reactions in proteomes.
Figure 2: Quantitative profiling of LDE-cysteine reactions in proteomes.
Figure 3: Determining the potency of HNE-cysteine reactions in proteomes and in cells.
Figure 4: Functional characterization of HNE modification of ZAK kinase.
Figure 5: HNE modification of ZAK suppresses JNK pathway activation in cells.

Accession codes

Accessions

Protein Data Bank

References

  1. 1

    Walsh, C.T. Posttranslational Modification of Proteins: Expanding Nature's Inventory (Roberts & Company, 2005).

  2. 2

    Jacobs, A.T. & Marnett, L.J. Systems analysis of protein modification and cellular responses induced by electrophile stress. Acc. Chem. Res. 43, 673–683 (2010).

    CAS  Article  Google Scholar 

  3. 3

    Leonard, S.E. & Carroll, K.S. Chemical 'omics' approaches for understanding protein cysteine oxidation in biology. Curr. Opin. Chem. Biol. 15, 88–102 (2011).

    CAS  Article  Google Scholar 

  4. 4

    Guéraud, F. et al. Chemistry and biochemistry of lipid peroxidation products. Free Radic. Res. 44, 1098–1124 (2010).

    Article  Google Scholar 

  5. 5

    Fritz, K.S. & Petersen, D.R. An overview of the chemistry and biology of reactive aldehydes. Free Radic. Biol. Med. 59, 85–91 (2013).

    CAS  Article  Google Scholar 

  6. 6

    Rudolph, T.K. & Freeman, B.A. Transduction of redox signaling by electrophile-protein reactions. Sci. Signal. 2, re7 (2009).

    Article  Google Scholar 

  7. 7

    Fritz, K.S. & Petersen, D.R. Exploring the biology of lipid peroxidation-derived protein carbonylation. Chem. Res. Toxicol. 24, 1411–1419 (2011).

    CAS  Article  Google Scholar 

  8. 8

    Leonarduzzi, G., Robbesyn, F. & Poli, G. Signaling kinases modulated by 4-hydroxynonenal. Free Radic. Biol. Med. 37, 1694–1702 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Jacobs, A.T. & Marnett, L.J. Heat shock factor 1 attenuates 4-Hydroxynonenal-mediated apoptosis: critical role for heat shock protein 70 induction and stabilization of Bcl-XL. J. Biol. Chem. 282, 33412–33420 (2007).

    CAS  Article  Google Scholar 

  10. 10

    Surh, Y.J. et al. 15-Deoxy-Δ12,14-prostaglandin J2, an electrophilic lipid mediator of anti-inflammatory and pro-resolving signaling. Biochem. Pharmacol. 82, 1335–1351 (2011).

    CAS  Article  Google Scholar 

  11. 11

    Chipuk, J.E. et al. Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis. Cell 148, 988–1000 (2012).

    CAS  Article  Google Scholar 

  12. 12

    Vila, A. et al. Identification of protein targets of 4-hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives. Chem. Res. Toxicol. 21, 432–444 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Codreanu, S.G., Zhang, B., Sobecki, S.M., Billheimer, D.D. & Liebler, D.C. Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal. Mol. Cell. Proteomics 8, 670–680 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Han, B., Hare, M., Wickramasekara, S., Fang, Y. & Maier, C.S. A comparative 'bottom up' proteomics strategy for the site-specific identification and quantification of protein modifications by electrophilic lipids. J. Proteomics 75, 5724–5733 (2012).

    CAS  Article  Google Scholar 

  15. 15

    Roe, M.R., Xie, H., Bandhakavi, S. & Griffin, T.J. Proteomic mapping of 4-hydroxynonenal protein modification sites by solid-phase hydrazide chemistry and mass spectrometry. Anal. Chem. 79, 3747–3756 (2007).

    CAS  Article  Google Scholar 

  16. 16

    Kim, H.Y., Tallman, K.A., Liebler, D.C. & Porter, N.A. An azido-biotin reagent for use in the isolation of protein adducts of lipid-derived electrophiles by streptavidin catch and photorelease. Mol. Cell Proteomics 8, 2080–2089 (2009).

    CAS  Article  Google Scholar 

  17. 17

    Aldini, G. et al. Identification of actin as a 15-deoxy-Δ12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement. Biochemistry 46, 2707–2718 (2007).

    CAS  Article  Google Scholar 

  18. 18

    Shearn, C.T., Fritz, K.S., Reigan, P. & Petersen, D.R. Modification of Akt2 by 4-hydroxynonenal inhibits insulin-dependent Akt signaling in HepG2 cells. Biochemistry 50, 3984–3996 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Bennaars-Eiden, A. et al. Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. J. Biol. Chem. 277, 50693–50702 (2002).

    CAS  Article  Google Scholar 

  20. 20

    Higdon, A.N. et al. Methods for imaging and detecting modification of proteins by reactive lipid species. Free Radic. Biol. Med. 47, 201–212 (2009).

    CAS  Article  Google Scholar 

  21. 21

    LoPachin, R.M., Gavin, T., Petersen, D.R. & Barber, D.S. Molecular mechanisms of 4-hydroxy-2-nonenal and acrolein toxicity: nucleophilic targets and adduct formation. Chem. Res. Toxicol. 22, 1499–1508 (2009).

    CAS  Article  Google Scholar 

  22. 22

    Doorn, J.A. & Petersen, D.R. Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2-nonenal and 4-oxo-2-nonenal. Chem. Res. Toxicol. 15, 1445–1450 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010).

    CAS  Article  Google Scholar 

  24. 24

    Rostovtsev, V.V., Green, J.G., Fokin, V.V. & Sharpless, K.B. A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Edn Engl. 41, 2596–2599 (2002).

    CAS  Article  Google Scholar 

  25. 25

    Yang, J.J. et al. ZAK inhibits human lung cancer cell growth via ERK and JNK activation in an AP-1-dependent manner. Cancer Sci. 101, 1374–1381 (2010).

    CAS  Article  Google Scholar 

  26. 26

    Bloem, L.J. et al. Tissue distribution and functional expression of a cDNA encoding a novel mixed lineage kinase. J. Mol. Cell Cardiol. 33, 1739–1750 (2001).

    CAS  Article  Google Scholar 

  27. 27

    Wong, J. et al. Small molecule kinase inhibitors block the ZAK-dependent inflammatory effects of doxorubicin. Cancer Biol. Ther. 14, 56–63 (2013).

    CAS  Article  Google Scholar 

  28. 28

    Wang, X. et al. Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA. J. Biol. Chem. 280, 19298–19305 (2005).

    CAS  Article  Google Scholar 

  29. 29

    Yu, X. & Bloem, L.J. Effect of C-terminal truncations on MLK7 catalytic activity and JNK activation. Biochem. Biophys. Res. Commun. 310, 452–457 (2003).

    CAS  Article  Google Scholar 

  30. 30

    Bachovchin, D.A. et al. Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors. Proc. Natl. Acad. Sci. USA 108, 6811–6816 (2011).

    CAS  Article  Google Scholar 

  31. 31

    Patricelli, M.P. et al. Functional interrogation of the kinome using nucleotide acyl phosphates. Biochemistry 46, 350–358 (2007).

    CAS  Article  Google Scholar 

  32. 32

    Shen, H.M. & Liu, Z.G. JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species. Free Radic. Biol. Med. 40, 928–939 (2006).

    CAS  Article  Google Scholar 

  33. 33

    Uchida, K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog. Lipid Res. 42, 318–343 (2003).

    CAS  Article  Google Scholar 

  34. 34

    Dubinina, E.E. & Dadali, V.A. Role of 4-hydroxy-trans-2-nonenal in cell functions. Biochemistry (Mosc.) 75, 1069–1087 (2010).

    CAS  Article  Google Scholar 

  35. 35

    Liu, Q. et al. Developing irreversible inhibitors of the protein kinase cysteinome. Chem. Biol. 20, 146–159 (2013).

    Article  Google Scholar 

  36. 36

    Huang, X. et al. Crystal structure of an inactive Akt2 kinase domain. Structure 11, 21–30 (2003).

    Article  Google Scholar 

  37. 37

    Wang, T., Kartika, R. & Spiegel, D.A. Exploring post-translational arginine modification using chemically synthesized methylglyoxal hydroimidazolones. J. Am. Chem. Soc. 134, 8958–8967 (2012).

    CAS  Article  Google Scholar 

  38. 38

    Weerapana, E., Simon, G.M. & Cravatt, B.F. Disparate proteome reactivity profiles of carbon electrophiles. Nat. Chem. Biol. 4, 405–407 (2008).

    CAS  Article  Google Scholar 

  39. 39

    Ban, H., Gavrilyuk, J. & Barbas, C.F. III. Tyrosine bioconjugation through aqueous ene-type reactions: a click-like reaction for tyrosine. J. Am. Chem. Soc. 132, 1523–1525 (2010).

    CAS  Article  Google Scholar 

  40. 40

    Eng, J.K., McCormack, A.L. & Yates, J.R. III. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

    CAS  Article  Google Scholar 

  41. 41

    Cociorva, D., Tabb, D.L. & Yates, J.R. Validation of tandem mass spectrometry database search results using DTASelect. Curr. Protoc. Bioinformatics 16, 13.4 (2007).

    Google Scholar 

  42. 42

    Patricelli, M.P. et al. In situ kinase profiling reveals functionally relevant properties of native kinases. Chem. Biol. 18, 699–710 (2011).

    CAS  Article  Google Scholar 

  43. 43

    Adibekian, A. et al. Click-generated triazole ureas as ultrapotent in vivo-active serine hydrolase inhibitors. Nat. Chem. Biol. 7, 469–478 (2011).

    CAS  Article  Google Scholar 

  44. 44

    Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank K. Backus, D. Bachovchin, B. Lanning, K. Tsuboi and A. Adibekian from the Cravatt Lab for providing reagents, and the Marletta lab at The Scripps Research Institute for sharing instrumentation for data collection. This work was supported by the US National Institutes of Health (NIH) (CA087660), an NIH/NIEHS K99/R00 Pathways to Independence Postdoctoral Award (K99ES020851, C.W.), a Pfizer Postdoctoral Fellowship (E.W.), a US National Science Foundation predoctoral fellowship (M.B.) and the Skaggs Institute for Chemical Biology.

Author information

Affiliations

Authors

Contributions

B.F.C., C.W. and E.W. conceived of the project. C.W., E.W. and M.M.B. performed experiments. B.F.C. and C.W. analyzed data and wrote the manuscript.

Corresponding authors

Correspondence to Chu Wang or Benjamin F Cravatt.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 771 kb)

Supplementary Table 1

In vitro profiling with each of the three LDEs at 100 μM in MDA-MB-231 and Ramos proteomes (XLS 6769 kb)

Supplementary Table 2

List of cysteines competed by one or more LDE with competition ratios > 5 (XLSX 51 kb)

Supplementary Table 3

In vitro profiling with HNE at 5 different concentrations in MDA-MB-231 proteomes (XLS 711 kb)

Supplementary Table 4

In situ profiling with HNE at 50 and 100 μM in MDA-MB-231 cells (XLS 422 kb)

Supplementary Table 5

List of kinases with reactive cysteines quantified by in vitro profiling with 100 μM HNE in MDA-MB-231 proteomes (XLSX 53 kb)

Supplementary Table 6

SILAC-ABPP ratios for acylphosphate-ATP probe-labeled proteins in proteomes from HEK-293T cells transfected with ZAK and treated with DMSO or 100 μM HNE in vitro (XLSX 1817 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wang, C., Weerapana, E., Blewett, M. et al. A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles. Nat Methods 11, 79–85 (2014). https://doi.org/10.1038/nmeth.2759

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing