The redox-switch domain of Hsp33 functions as dual stress sensor

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Abstract

The redox-regulated chaperone Hsp33 is specifically activated upon exposure of cells to peroxide stress at elevated temperatures. Here we show that Hsp33 harbors two interdependent stress-sensing regions located in the C-terminal redox-switch domain of Hsp33: a zinc center sensing peroxide stress conditions and an adjacent linker region responding to unfolding conditions. Neither of these sensors works sufficiently in the absence of the other, making the simultaneous presence of both stress conditions a necessary requirement for Hsp33's full activation. Upon activation, Hsp33's redox-switch domain adopts a natively unfolded conformation, thereby exposing hydrophobic surfaces in its N-terminal substrate-binding domain. The specific activation of Hsp33 by the oxidative unfolding of its redox-switch domain makes this chaperone optimally suited to quickly respond to oxidative stress conditions that lead to protein unfolding.

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Figure 1: Domain structure of Hsp33.
Figure 2: In vitro activation of Hsp33 requires oxidative stress at elevated temperatures.
Figure 3: Thiol status of Hsp33's tryptic peptide 232-CTCSR-236 (calculated m/z = 569.7), containing the first redox-active cysteine pair.
Figure 4: Activation of Hsp33 is accompanied by major conformational rearrangements.
Figure 5: C-terminal truncation mutant Hsp331–235 functions as a redox-regulated chaperone.
Figure 6: Unfolding of the linker region is crucial for Hsp33's activation.
Figure 7: Schematic model of Hsp33's activation process.

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Acknowledgements

We thank S. VanHaerents for excellent technical assistance, J. Bardwell, L. Leichert and T. Tapley for critically reading the manuscript, and Leopoldina Gesellschaft Deutscher Naturforscher for a postdoctoral fellowship to J.W. P.C.F.G. was supported by a Rackham Predoctoral Fellowship. US National Institutes of Health grant GM065318 supported this work.

Author information

M.I. performed the majority of the experiments, including the activation studies, zinc-release analysis, mass spectrometry, fluorescence spectroscopy and Gdn-HCl experiments. J.H. constructed the mutant proteins and performed the fluorescence experiments. S.A. analyzed the Hsp331–235 mutant protein. J.W. performed the two-dimensional gel analysis. P.C.F.G. performed the thermostability experiments and initiated many of the experiments. H.L. conducted the ultracentrifugation experiments and contributed to data evaluation. U.J. contributed ideas, evaluated and discussed data and prepared the manuscript.

Correspondence to Ursula Jakob.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

AMS trapping reveals difference in thiol modification patterns of Hsp33 incubated in H2O2 at 30 °C or 43 °C. (PDF 59 kb)

Supplementary Fig. 2

Hsp33's C terminus is predicted to be natively unfolded in the absence of cofactors. (PDF 35 kb)

Supplementary Fig. 3

Peroxide stress does not induce protein aggregation in vivo. (PDF 323 kb)

Supplementary Table 1

α-helix content in reduced and oxidized Hsp33. (PDF 115 kb)

Supplementary Data (PDF 73 kb)

Supplementary Methods (PDF 23 kb)

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Ilbert, M., Horst, J., Ahrens, S. et al. The redox-switch domain of Hsp33 functions as dual stress sensor. Nat Struct Mol Biol 14, 556–563 (2007) doi:10.1038/nsmb1244

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