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

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.Reactive oxygen species develop as unavoidable consequences of aerobic life. Their oxidizing effects on most cellular macromolecules can be deleterious to cells and organisms 1 . Cells have developed effective antioxidant systems to counteract this hazard (for review, see ref. 2 ). Disruption of the fine balance between oxidants and antioxidants, however, leads to the accumulation of reactive oxygen species and to a condition termed oxidative stress 3 . Oxidative stress conditions have been shown to develop in, and may even cause, numerous physiological and pathological conditions, such as aging, heart disease, diabetes and neurodegenerative diseases 4 .

Quantifying changes in the thiol redox proteome upon oxidative stress in vivo

“…Oxidative stress-induced formation of two intramolecular disulfide bonds converts reduced, inactive Hsp33 into a potent chaperone that protects bacteria against oxidative stress (19). Recent studies suggested that although one disulfide bond in Hsp33 forms very quickly, formation of the other disulfide bond (Cys-232-Cys-234) is slow and might represent the rate-determining step in Hsp33's activation (20). However, appropriate quantitative methods to test this model were not available.…”

“…To monitor the kinetics of thiol modifications during Hsp33's activation process, we incubated reduced Hsp33 with H 2 O 2 at 43°C (20). At defined time points, we removed aliquots and either tested them for chaperone activity or used them for our OxICAT labeling.…”

“…Reduced and oxidized Hsp33 was prepared as described (20). To activate Hsp33, reduced Hsp33 was incubated with 2 mM H 2O2 for 1 h at 43°C.…”

“…Aliquots were removed and the influence of Hsp33 on the aggregation of chemically unfolded citrate synthase at 30°C was assayed as described in ref. 20. For OxICAT analysis, proteins were precipitated with 10% (wt/vol) TCA.…”

Redox Biology: Computational Approaches to the Investigation of Functional Cysteine Residues

“…In this regard, Cys properties make this amino acid a preferred residue for redoxdependent regulation of metal binding. For example, the Zn 2þ -S moiety permits zinc to be tightly bound, yet be available for release upon oxidation (41,43,44,50). We…”