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.

Firefly luciferase mutants as sensors of proteome stress

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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

References

  1. Powers, E.T., Morimoto, R.I., Dillin, A., Kelly, J.W. & Balch, W.E. Biological and chemical approaches to diseases of proteostasis deficiency. Annu. Rev. Biochem. 78, 959–991 (2009).

    CAS  Article  Google Scholar 

  2. Vabulas, R.M., Raychaudhuri, S., Hayer-Hartl, M. & Hartl, F.U. Protein folding in the cytoplasm and the heat shock response. Cold Spring Harb. Perspect. Biol. 2, a004390 (2010).

    CAS  Article  Google Scholar 

  3. Frydman, J. Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu. Rev. Biochem. 70, 603–647 (2001).

    CAS  Article  Google Scholar 

  4. Rubinsztein, D.C. The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 443, 780–786 (2006).

    CAS  Article  Google Scholar 

  5. Ben-Zvi, A., Miller, E.A. & Morimoto, R.I. Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging. Proc. Natl. Acad. Sci. USA 106, 14914–14919 (2009).

    CAS  Article  Google Scholar 

  6. Balch, W.E., Morimoto, R.I., Dillin, A. & Kelly, J.W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008).

    CAS  Article  Google Scholar 

  7. Mu, T.W. et al. Chemical and biological approaches synergize to ameliorate protein-folding diseases. Cell 134, 769–781 (2008).

    CAS  Article  Google Scholar 

  8. Gidalevitz, T., Ben-Zvi, A., Ho, K.H., Brignull, H.R. & Morimoto, R.I. Progressive disruption of cellular protein folding in models of polyglutamine diseases. Science 311, 1471–1474 (2006).

    CAS  Article  Google Scholar 

  9. Frydman, J., Nimmesgern, E., Ohtsuka, K. & Hartl, F.U. Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature 370, 111–117 (1994).

    CAS  Article  Google Scholar 

  10. Thulasiraman, V. & Matts, R.L. Effect of geldanamycin on the kinetics of chaperone-mediated renaturation of firefly luciferase in rabbit reticulocyte lysate. Biochemistry 35, 13443–13450 (1996).

    CAS  Article  Google Scholar 

  11. Nimmesgern, E. & Hartl, F.U. ATP-dependent protein refolding activity in reticulocyte lysate. Evidence for the participation of different chaperone components. FEBS Lett. 331, 25–30 (1993).

    CAS  Article  Google Scholar 

  12. Schroder, H., Langer, T., Hartl, F.U. & Bukau, B. DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J. 12, 4137–4144 (1993).

    CAS  Article  Google Scholar 

  13. Conti, E., Franks, N.P. & Brick, P. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 4, 287–298 (1996).

    CAS  Article  Google Scholar 

  14. Naylor, L.H. Reporter gene technology: the future looks bright. Biochem. Pharmacol. 58, 749–757 (1999).

    CAS  Article  Google Scholar 

  15. Hageman, J., Vos, M.J., van Waarde, M.A. & Kampinga, H.H. Comparison of intra-organellar chaperone capacity for dealing with stress-induced protein unfolding. J. Biol. Chem. 282, 34334–34345 (2007).

    CAS  Article  Google Scholar 

  16. Michels, A.A., Nguyen, V.T., Konings, A.W., Kampinga, H.H. & Bensaude, O. Thermostability of a nuclear-targeted luciferase expressed in mammalian cells. Destabilizing influence of the intranuclear microenvironment. Eur. J. Biochem. 234, 382–389 (1995).

    CAS  Article  Google Scholar 

  17. Matsui, I. & Harata, K. Implication for buried polar contacts and ion pairs in hyperthermostable enzymes. FEBS J. 274, 4012–4022 (2007).

    CAS  Article  Google Scholar 

  18. Schneider, C. et al. Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90. Proc. Natl. Acad. Sci. USA 93, 14536–14541 (1996).

    CAS  Article  Google Scholar 

  19. Sharp, S. & Workman, P. Inhibitors of the HSP90 molecular chaperone: current status. Adv. Cancer Res. 95, 323–348 (2006).

    CAS  Article  Google Scholar 

  20. Taipale, M., Jarosz, D.F. & Lindquist, S. HSP90 at the hub of protein homeostasis: emerging mechanistic insights. Nat. Rev. Mol. Cell Biol. 11, 515–528 (2010).

    CAS  Article  Google Scholar 

  21. Muchowski, P.J. Protein misfolding, amyloid formation, and neurodegeneration: a critical role for molecular chaperones? Neuron 35, 9–12 (2002).

    CAS  Article  Google Scholar 

  22. Broadley, S.A. & Hartl, F.U. The role of molecular chaperones in human misfolding diseases. FEBS Lett. 583, 2647–2653 (2009).

    CAS  Article  Google Scholar 

  23. Morimoto, R.I. & Cuervo, A.M. Protein homeostasis and aging: taking care of proteins from the cradle to the grave. J. Gerontol. A Biol. Sci. Med. Sci. 64, 167–170 (2009).

    Article  Google Scholar 

  24. Kern, A., Ackermann, B., Clement, A.M., Duerk, H. & Behl, C. HSF1-controlled and age-associated chaperone capacity in neurons and muscle cells of C. elegans. PLoS ONE 5, e8568 (2010).

    Article  Google Scholar 

  25. Kaganovich, D., Kopito, R. & Frydman, J. Misfolded proteins partition between two distinct quality control compartments. Nature 454, 1088–1095 (2008).

    CAS  Article  Google Scholar 

  26. Nakatsu, T. et al. Structural basis for the spectral difference in luciferase bioluminescence. Nature 440, 372–376 (2006).

    CAS  Article  Google Scholar 

  27. Behrends, C. et al. Chaperonin TRiC promotes the assembly of polyQ expansion proteins into nontoxic oligomers. Mol. Cell 23, 887–897 (2006).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

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.

Ethics declarations

Competing interests

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

Supplementary information

Supplementary Text and Figures

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

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1697

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