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Unraveling the mechanism of cell death induced by chemical fibrils

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Abstract

We previously discovered a small-molecule inducer of cell death, named 1541, that noncovalently self-assembles into chemical fibrils ('chemi-fibrils') and activates procaspase-3 in vitro. We report here that 1541-induced cell death is caused by the fibrillar rather than the soluble form of the drug. A short hairpin RNA screen reveals that knockdown of genes involved in endocytosis, vesicle trafficking and lysosomal acidification causes partial 1541 resistance. We confirm the role of these pathways using pharmacological inhibitors. Microscopy shows that the fluorescent chemi-fibrils accumulate in punctae inside cells that partially colocalize with lysosomes. Notably, the chemi-fibrils bind and induce liposome leakage in vitro, suggesting they may do the same in cells. The chemi-fibrils induce extensive proteolysis including caspase substrates, yet modulatory profiling reveals that chemi-fibrils form a distinct class from existing inducers of cell death. The chemi-fibrils share similarities with proteinaceous fibrils and may provide insight into their mechanism of cellular toxicity.

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Figure 1: 1541 forms chemi-fibrils that induce cell death in cell culture.
Figure 2: shRNA screen reveals a major role of vesicle trafficking and lysosome acidification in 1541 chemi-fibril–induced cell death.
Figure 3: Internalization of fluorescent aggregates in cell and specific endocytosis inhibition delay cell death.
Figure 4: Cell death induced by chemi-fibrils induces proteolysis with prominent caspase cleavages.
Figure 5: Modulatory profiling suggests 1541 and an analog form a unique class of compounds that induce cell death only partially delayed by blocking caspases.
Figure 6: Proposed mechanism of cell death induced by 1541 chemi-fibrils.

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Acknowledgements

We would like to thank F. Brodsky, B. Shoichet, W. Degrado, M. Zhuang, A. Wiita, Z. Hill, J.T. Koerber, N. Thomsen, J. Watts, S.-A. Mok and J. Rettenmaier for insightful discussions and/or critical reading of the manuscript. A special thanks to Y. Chen (cell culture and laboratory practices expertise), Y. Cheng and M. Braunfield (EM), A. Doak (DLS), H. Tran (yeast expertise), D. Larsen (live cell imaging), J. Lund (deep sequencing), M. Hornsby and K. Verba (fluorescence) and T. Matsuguchi (qPCR) for technical help. This work was supported, in whole or in part, by US National Institutes of Health grant R01 CA136779 (to J.A.W.), R01 CA097061 (to B.R.S.) and F32AI095062 (to V.J.V.) and by the Howard Hughes Medical Institute (to B.R.S. and J.S.W.). J.A.Z. received an Achievement Rewards for College Scientists Foundation Award and a Schleroderma Research Foundation Evnin-Wright Fellowship. M.K. was supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund. O.J. is the recipient of a Banting Postdoctoral Fellowship funded by the Canadian Institutes of Health Research and the Government of Canada. O.J. and M.K. both received a fellowship from the University of California–San Francisco Program for Breakthrough Biomedical Research, which is funded in part by the Sandler Foundation.

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Contributions

O.J. performed the cell culture experiments, EM, DLS, flow cytometry and fluorescence microscopy under J.A.W.'s supervision. O.J. performed the shRNA screen and made the stable cell lines, with help and guidance from M.K. and M.C.B. and supervision of J.S.W.; M.K. analyzed the deep-sequencing data. J.A.Z. synthesized the 1541 analogs and provided general expertise on the project. A.L.R. performed the crystallography and structure determination. V.J.V. performed the liposome leakage assays. K. Shimbo and N.J.A. performed the degradomics experiments, and O.J. compiled the results. K. Shimada performed the modulatory profiling experiments under B.R.S.'s supervision. O.J. and J.A.W. wrote the manuscript with contributions from M.K., J.A.Z. and B.R.S., with input from all authors.

Corresponding author

Correspondence to James A Wells.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results and Supplementary Figures 1–22. (PDF 12319 kb)

Supplementary Data Set 1

P values for all genes tested in the shRNA screen. (XLSX 324 kb)

Supplementary Data Set 2

Identification of proteolytic fragments generated during chemi-fibril induced cell death. (XLSX 3535 kb)

Supplementary Video 1

Live cell imaging of HeLa cells treated with chemi-fibrils. (MOV 5773 kb)

Supplementary Video 2

Live cell imaging of HeLa cells treated with chemi-fibrils. (MOV 13992 kb)

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Julien, O., Kampmann, M., Bassik, M. et al. Unraveling the mechanism of cell death induced by chemical fibrils. Nat Chem Biol 10, 969–976 (2014). https://doi.org/10.1038/nchembio.1639

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