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Redox-Regulated Molecular Chaperones

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Redox-Regulated Molecular Chaperones CMLS, Cell. Mol. Life Sci. 59 (2002) 1624–1631 1420-682X/02/101624-08 © Birkhäuser Verlag, Basel, 2002 CMLS Cellular and Molecular Life Sciences Redox-regulated molecular chaperones P. C . F. G ra f b and U. Jakob a,b,* a Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 N. University Avenue, Ann Arbor, Michigan 48109-1048 (USA), Fax +1 734 647 0884, e-mail: [email protected] b Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, Michigan 48109-1048 (USA) Abstract. The conserved heat shock protein Hsp33 func- bonds under oxidizing, activating conditions. Hsp33’s re- tions as a potent molecular chaperone with a highly so- dox-regulated chaperone activity appears to specifically phisticated regulation. On transcriptional level, the protect proteins and cells from the otherwise deleterious Hsp33 gene is under heat shock control; on posttransla- effects of reactive oxygen species. That redox regulation tional level, the Hsp33 protein is under oxidative stress of chaperone activity is not restricted to Hsp33 became control. This dual regulation appears to reflect the close evident when the chaperone activity of protein disulfide but rather neglected connection between heat shock and isomerase was recently also shown to cycle between a oxidative stress. The redox sensor in Hsp33 is a cysteine low- and high-affinity substrate binding state, depending center that coordinates zinc under reducing, inactivating on the redox state of its cysteines. conditions and that forms two intramolecular disulfide Key words. Hsp33; PDI; redox regulation; disulfide bond formation; oxidative stress; heat shock; metal centers; re- active oxygen species. Hsp33-A chaperone on the interface of oxidative twice the level of Hsp33 under nonstress conditions [5]. stress and heat shock This is similar to the concentration reported for the double-doughnut chaperonin GroEL (2.6 mM) [6]. Hsp33 is a highly conserved heat shock protein that is pre- Thus, on transcriptional level, the Hsp33 gene is under sent in the cytosol of more than 50 different prokaryotic heat shock control; on posttranslational level, the Hsp33 organisms. Recently, the first eukaryotic Hsp33 homo- protein is under oxidative stress control [5]. This post- logue was identified in the unicellular photosynthetic or- translational mode of regulation enables the Hsp33 pro- ganism Chlamydomonas reinhardtii, where it is predicted tein to sense changes in the redox conditions of the envi- to be localized to the chloroplasts by PSORT [1]. ronment and to translate these changes into differences in The transcription of hslO (originally named for heat protein conformation and chaperone activity. Hsp33’s re- shock locus O), the gene encoding Hsp33, is under heat dox sensor is located in the C-terminus of the protein. It shock control [2]. In Escherichia coli, hslO forms part of consists of four highly conserved cysteines that coordi- a heat-shock-regulated multigene operon. It is located nate zinc in the reduced, low-affinity substrate-binding downstream of hslR, a gene encoding the 50S ribosomal conformation, and that form two intramolecular disulfide binding protein Hsp15 [3], and hslP, a gene encoding a bonds in the oxidized, high-affinity substrate-binding putative heat-shock-regulated phosphatase. Upon expo- conformation of Hsp33 [5]. The redox potential of Hsp sure of E. coli to heat shock conditions, the messenger 33’s chaperone activity has been shown to be –170 mV RNA (mRNA) level of the Hsp33 gene increases about [5]. This implies that under nonstress conditions, where 30-fold, while the mRNA level of hslR and hslP increase the bacterial cytosol is thought to be strongly reducing about 40- and 20-fold, respectively [4]. This increases the (–250 to –280 mV) [7], Hsp33’s cysteines coordinate steady-state protein concentration of Hsp33 to ~3 mM, zinc, and the protein is mostly deactivated. Under oxi- dative stress conditions, on the other hand, where the cel- * Corresponding author. lular redox potential can increase to values of –150 mV CMLS, Cell. Mol. Life Sci. Vol. 59, 2002 Multi-author Review Article 1625 or more [7], Hsp33’s cysteines become oxidized, and are in their highly reactive thiolate anion state when the the protein is activated [5] (also reviewed by [8, 9]). The metal center is occupied. This may be due to the zinc(II) redox potential of Hsp33 is very similar to the redox po- cation acting as a Lewis acid that can stabilize the nega- tential of OxyR, the oxidative stress transcription factor tive charges on the thiolate anions, thereby lowering their in E. coli (–185 mV) [10]. OxyR is preferentially reduced pKa values and making them more reactive to hydrogen and inactive under normal conditions, but becomes peroxide at near neutral pH [20]. This thiolate anion state rapidly activated upon exposure of the cells to H2O2 in combination with the close proximity of the thiol [10–12]. Similar to Hsp33, OxyR’s activation mecha- groups in the tertiary structure has been postulated to nism involves the formation of an intramolecular disul- prime the cysteines for a rapid reaction to form disulfide fide bond. Noteworthy, Hsp33’s transcription is not under bonds [15]. OxyR control [13, 14]. Hsp33’s activation on a posttranslational level under ox- idative stress will lead to a certain level of chaperone ac- Activation: a two-step process tivity that might be sufficient for the cells as a first line of defense against agents that cause oxidative damage. Biochemical studies revealed that Hsp33’s activation When the oxidative stress conditions lead to such severe process occurs in two steps [19]. The first step involves protein damage that the heat shock response is induced, formation of two intramolecular disulfide bonds and con- Hsp33’s concentration will also increase. current release of zinc. This step is independent of Hsp33 protein concentration and occurs prior to the full activa- tion of Hsp33, a process that involves dimerization of two The on-off switch: a cysteine coordinating oxidized Hsp33 monomers. This second step is both zinc center highly temperature and concentration dependent, optimal for a protein that is overexpressed under heat shock con- Hsp33 from E. coli has six cysteine residues, four of ditions. Oxidized and fully active Hsp33 dimer has a KD which are highly conserved. These four conserved cys- of 0.6 mM at 20°C [19]. Since the dimerization of oxi- teines, Cys-X-Cys-X27–32-Cys-X-X-Cys, form a novel, dized Hsp33 appears to be dependent on hydrophobic in- very high affinity zinc binding motif [15]. The Ka of re- teractions between both monomers at the dimer interface 17 –1 duced Hsp33 for zinc(II) at 25°C, pH 7.5 is 2.5 ¥ 10 M [19], elevated temperatures should lower the KD, favor the [15]. This indicates a stronger interaction between zinc dimerization process and significantly accelerate the ac- and Hsp33 than between zinc and the cytosolic zinc tivation of Hsp33 under heat shock conditions. Gel filtra- storage protein, metallothionein, which has a Ka of 3.2 ¥ tion studies with a constitutively active Hsp33 truncation 1013 M–1, pH 7.4 [16, 17] and many other zinc-binding mutant (aa 1–235) confirmed the presence of dimers at proteins. The zinc ion appears to be coordinated by all physiological Hsp33 concentrations. At lower Hsp33 con- four conserved cysteines in a tetrahedral geometry, as ev- centrations, addition of denatured protein substrates has idenced by metal replacement studies with cobalt(II) been shown to shift the equilibrium of the oxidized trun- [15]. The zinc center in Hsp33 folds in a very stable do- cation mutant toward the dimeric conformation [21]. main that is highly protease resistant when compared Extensive conformational changes appear to accompany with zinc-free reduced Hsp33 [15]. Not even the unfold- the activation process of Hsp33. Far ultraviolet (UV)-cir- ing of Hsp33 in 6 M guanidine hydrochloride causes the cular dichroism spectra revealed that the oxidation of re- release of the bound zinc [15]. In contrast, exposure of duced Hsp33 is accompanied by significant changes in its Hsp33 to oxidizing conditions (e.g. H2O2) releases the secondary structure. Based on this analysis, reduced zinc immediately. This appears to be due to the formation Hsp33 is predicted to contain ~31% random coil, 44% a of two intramolecular disulfide bonds, which connect the helix and 25% b sheet, while the oxidized, active Hsp33 two next-neighbor cysteines Cys232 with Cys234 and Cys265 revealed significantly increased random coil (49%) at the with Cys268 [18]. This oxidation induced disulfide bond expense of a helix (35%) [22]. A substantial increase in formation and zinc release is the first step in the activa- hydrophobic surfaces has been shown to accompany the tion process of Hsp33 [19]. activation process of Hsp33 [22]. This may be important Despite the fact that zinc is a redox inert metal, experi- in Hsp33’s chaperone function. Using the fluorescence ments with zinc-reconstituted and zinc-free reduced properties of the small hydrophobic dye bis-ANS, it has Hsp33 have shown that zinc plays a rather active role in been demonstrated that while reduced Hsp33 barely in- the activation process of Hsp33. Zinc-free Hsp33 reacti- teracts with the hydrophobic probe, oxidized Hsp33 vates slowly and incompletely when exposed to oxidative shows a major blue-shifted fluorescence signal [22]. This stress treatment in vitro, whereas the reactivation of zinc- suggests that substantial rearrangements occur upon oxi- coordinated Hsp33 occurs quickly and goes to comple- dation and dimerization of Hsp33 that allow the forma- tion [15]. We have shown that all four cysteines of Hsp33 tion or exposure of a predominantly hydrophobic sub- 1626 P. C. F. Graf and U. Jakob Redox-regulated molecular chaperones strate binding site that is either nonexistent or buried in productive refolding of the substrate proteins, once the the reduced, inactive Hsp33 monomer.
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