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A reactivity-based probe of the intracellular labile ferrous iron pool

Nature Chemical Biology volume 12, pages 680–685 (2016)Cite this article

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Abstract

Improved methods for studying intracellular reactive Fe(II) are of significant interest for studies of iron metabolism and disease-relevant changes in iron homeostasis. Here we describe a highly selective reactivity-based probe in which a Fenton-type reaction with intracellular labile Fe(II) leads to unmasking of the aminonucleoside puromycin. Puromycin leaves a permanent and dose-dependent mark on treated cells that can be detected with high sensitivity and precision using a high-content, plate-based immunofluorescence assay. Using this new probe and screening approach, we detected alteration of cellular labile Fe(II) in response extracellular iron conditioning, overexpression of iron storage and/or export proteins, and post-translational regulation of iron export. We also used this new tool to demonstrate that labile Fe(II) pools are larger in cancer cells than in nontumorigenic cells.

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Figure 1: A turn-on fluorescent probe reveals Fe(II)-selective reactivity of trioxolanes.
Figure 2: Trioxolane conjugate of puromycin as a cellular probe of Fe(II).
Figure 3: Reactivity-based probe 3 is highly selective for Fe(II) in cells.
Figure 4: Comparing reactive Fe(II) pools across cell lines and genetic modulations.

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Acknowledgements

The authors thank S. Chen for technical support in automated cell imaging and image analysis with IN Cell Developer software, T. Matsuguchi for assistance with qRT-PCR setup and analysis, H. Shimizu for assistance with the PAMPA assay, and D. Ruggero for helpful comments on the manuscript. A.R.R. is funded by US National Institutes of Health (NIH) grant AI105106. C.J.C. is funded by NIH grant GM 79465. B.S. and C.W.M. acknowledge funding from the NIH Research Training Grant in Chemistry and Chemical Biology (T32 GM064337).

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Authors and Affiliations

  1. Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, California, USA

    Benjamin Spangler, Charles W Morgan & Shaun D Fontaine

  2. Department of Chemistry, University of California, Berkeley, Berkeley, California, USA

    Mark N Vander Wal & Christopher J Chang

  3. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA

    Mark N Vander Wal & Christopher J Chang

  4. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA

    Christopher J Chang

  5. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA

    James A Wells & Adam R Renslo

  6. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA

    James A Wells

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Contributions

B.S., J.A.W., and A.R.R. conceived and designed experiments. B.S., C.W.M. and S.D.F. carried out experiments. B.S., C.W.M., S.D.F., J.A.W., and A.R.R. analyzed data. B.S., C.W.M., and A.R.R. wrote the manuscript. M.N.V.W. and C.J.C. provided reagents and discussion. All authors read and commented on the manuscript.

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Correspondence to James A Wells or Adam R Renslo.

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Competing interests

B.S., S.D.F., J.A.W., and A.R.R. are listed as inventors on a patent application describing 3 and related trioxolane conjugates.

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Supplementary Results, Supplementary Figures 1–9 and Supplementary Table 1. (PDF 2897 kb)

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Spangler, B., Morgan, C., Fontaine, S. et al. A reactivity-based probe of the intracellular labile ferrous iron pool. Nat Chem Biol 12, 680–685 (2016). https://doi.org/10.1038/nchembio.2116

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