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Firefly luciferase mutants as sensors of proteome stress

Nature Methods volume 8, pages 879–884 (2011)Cite this article

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Abstract

Maintenance of cellular protein homeostasis (proteostasis) depends on a complex network of molecular chaperones, proteases and other regulatory factors. Proteostasis deficiency develops during normal aging and predisposes individuals for many diseases, including neurodegenerative disorders. Here we describe sensor proteins for the comparative measurement of proteostasis capacity in different cell types and model organisms. These sensors are increasingly structurally destabilized versions of firefly luciferase. Imbalances in proteostasis manifest as changes in sensor solubility and luminescence activity. We used EGFP-tagged constructs to monitor the aggregation state of the sensors and the ability of cells to solubilize or degrade the aggregated proteins. A set of three sensor proteins serves as a convenient toolkit to assess the proteostasis status in a wide range of experimental systems, including cell and organism models of stress, neurodegenerative disease and aging.

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Figure 1: Thermal stability of luciferase sensor proteins.
Figure 2: Chaperone dependence and thermal stability of sensor proteins in HeLa cells.
Figure 3: Effect of Fluc-based sensors on the cytosolic stress response.
Figure 4: Fluc-based sensors report on proteostasis impairment by small-molecule inhibitors or neurodegenerative disease protein.
Figure 5: Fluc-based sensors report on acute proteome stress during heat shock in C. elegans.
Figure 6: Fluc-based sensors report on proteostasis decline during aging in C. elegans.

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Acknowledgements

We thank M. Hipp, Y.E. Kim and M. Hayer-Hartl for discussion, F. Buchholz (Max Planck Institute for Molecular Cell Biology and Genetics) for providing Hsc70 endoribonuclease-prepared small interfering (esi)RNA, and H. Wagner (Institut für Med. Mikrobiologie, Immunologie und Hygiene, Technische Universität München) for the HSP70-Luc plasmid. We acknowledge the expert technical assistance of V. Marcus. This work has been supported by the European Commission within the 7th framework program Proteomics Specification in Time and Space.

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Authors and Affiliations

  1. Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany

    Rajat Gupta, Prasad Kasturi, Andreas Bracher, Christian Loew, Min Zheng, F Ulrich Hartl & Swasti Raychaudhuri

  2. Proteostasis Therapeutics Inc., Cambridge, Massachusetts, USA

    Adriana Villella & Dan Garza

  3. Center for Integrated Protein Science, München, Germany

    F Ulrich Hartl

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  1. Rajat Gupta
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Contributions

S.R. and F.U.H. conceived the idea and developed the method. A.B. designed Fluc mutants. R.G., C.L. and S.R. performed molecular biology and cell biology experiments. P.K. and M.Z. performed C. elegans experiments. A.V. and D.G. performed D. melanogaster S2 cell experiments. R.G. and S.R. analyzed results. S.R. and F.U.H. interpreted results and wrote the manuscript with assistance from D.G.

Corresponding authors

Correspondence to F Ulrich Hartl or Swasti Raychaudhuri.

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Competing interests

F.U.H is a paid consultant of Proteostasis Therapeutics Inc. A.V and D.G are employees of Proteostasis Therapeutics Inc.

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Supplementary Figures 1–8, Supplementary Tables 1–3, Supplementary Note 1 (PDF 5904 kb)

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Gupta, R., Kasturi, P., Bracher, A. et al. Firefly luciferase mutants as sensors of proteome stress. Nat Methods 8, 879–884 (2011). https://doi.org/10.1038/nmeth.1697

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