Reactive oxygen species accumulation is synchronised with growth inhibition of temperature-sensitive recAts polA Escherichia coli.

When combined with recombinase defects, chromosome breakage and double-strand break repair deficiencies render cells inviable. However, cells are viable when an SOS response occurs in recAts polA cells in Escherichia coli. Here, we aimed to elucidate the underlying mechanisms of this process. Transposon mutagenesis revealed that the hslO gene, a redox chaperone Hsp33 involved in reactive oxidative species (ROS) metabolism, was required for the suppression of recAts polA lethality at a restricted temperature. Recently, it has been reported that lethal treatments trigger ROS accumulation. We also found that recAts polA cells accumulated ROS at the restricted temperature. A catalase addition to the medium alleviates the temperature sensitivity of recAts polA cells and decreases ROS accumulation. These results suggest that the SOS response and hslO manage oxidative insult to an acceptable level in cells with oxidative damage and rescue cell growth. Overall, ROS might regulate several cellular processes.

Molecular Effects of Elongation Factor Ts and Trigger Factor on the Unfolding and Aggregation of Elongation Factor Tu Induced by the Prokaryotic Molecular Chaperone Hsp33.

Hsp33, a prokaryotic redox-regulated holding chaperone, has been recently identified to be able to exhibit an unfoldase and aggregase activity against elongation factor Tu (EF-Tu) in its reduced state. In this study, we investigated the effect of elongation factor Ts (EF-Ts) and trigger factor (TF) on Hsp33-mediated EF-Tu unfolding and aggregation using gel filtration, light scattering, circular dichroism, and isothermal titration calorimetry. We found that EF-Tu unfolding and subsequent aggregation induced by Hsp33 were evident even in its complex state with EF-Ts, which enhanced EF-Tu stability. In addition, although TF alone had no substantial effect on the stability of EF-Tu, it markedly amplified the Hsp33-mediated EF-Tu unfolding and aggregation. Collectively, the present results constitute the first example of synergistic unfoldase/aggregase activity of molecular chaperones and suggest that the stability of EF-Tu is modulated by a sophisticated network of molecular chaperones to regulate protein biosynthesis in cells under stress conditions.

The Central Role of Redox-Regulated Switch Proteins in Bacteria.

Bacteria possess the ability to adapt to changing environments. To enable this, cells use reversible post-translational modifications on key proteins to modulate their behavior, metabolism, defense mechanisms and adaptation of bacteria to stress. In this review, we focus on bacterial protein switches that are activated during exposure to oxidative stress. Such protein switches are triggered by either exogenous reactive oxygen species (ROS) or endogenous ROS generated as by-products of the aerobic lifestyle. Both thiol switches and metal centers have been shown to be the primary targets of ROS. Cells take advantage of such reactivity to use these reactive sites as redox sensors to detect and combat oxidative stress conditions. This in turn may induce expression of genes involved in antioxidant strategies and thus protect the proteome against stress conditions. We further describe the well-characterized mechanism of selected proteins that are regulated by redox switches. We highlight the diversity of mechanisms and functions (as well as common features) across different switches, while also presenting integrative methodologies used in discovering new members of this family. Finally, we point to future challenges in this field, both in uncovering new types of switches, as well as defining novel additional functions.

TrypOx, a Novel Eukaryotic Homolog of the Redox-Regulated Chaperone Hsp33 in Trypanosoma brucei.

ATP-independent chaperones are widespread across all domains of life and serve as the first line of defense during protein unfolding stresses. One of the known crucial chaperones for bacterial survival in a hostile environment (e.g., heat and oxidative stress) is the highly conserved, redox-regulated ATP-independent bacterial chaperone Hsp33. Using a bioinformatic analysis, we describe novel eukaryotic homologs of Hsp33 identified in eukaryotic pathogens belonging to the kinetoplastids, a family responsible for lethal human diseases such as Chagas disease as caused by Trypanosoma cruzi, African sleeping sickness caused by Trypanosoma brucei spp., and leishmaniasis pathologies delivered by various Leishmania species. During their pathogenic life cycle, kinetoplastids need to cope with elevated temperatures and oxidative stress, the same conditions which convert Hsp33 into a powerful chaperone in bacteria, thus preventing aggregation of a wide range of misfolded proteins. Here, we focused on a functional characterization of the Hsp33 homolog in one of the members of the kinetoplastid family, T. brucei, (Tb927.6.2630), which we have named TrypOx. RNAi silencing of TrypOx led to a significant decrease in the survival of T. brucei under mild oxidative stress conditions, implying a protective role of TrypOx during the Trypanosomes growth. We then adopted a proteomics-driven approach to investigate the role of TrypOx in defining the oxidative stress response. Depletion of TrypOx significantly altered the abundance of proteins mediating redox homeostasis, linking TrypOx with the antioxidant system. Using biochemical approaches, we identified the redox-switch domain of TrypOx, showing its modularity and oxidation-dependent structural plasticity. Kinetoplastid parasites such as T. brucei need to cope with high levels of oxidants produced by the innate immune system, such that parasite-specific antioxidant proteins like TrypOx - which are depleted in mammals - are highly promising candidates for drug targeting.

Immune response and protective effect against Vibrio anguillarum induced by DNA vaccine encoding Hsp33 protein.

Heat shock proteins (HSPs) are considered as potent vaccine candidates against a wide range of bacterial diseases. In the present study, a recombinant DNA plasmid based on the expression of Hsp33 gene was constructed and intramuscularly administrated to flounder to elucidate whether it induces immune response and prevents the infection of Vibrio anguillarum in flounder model. Meanwhile, the expression of pHsp33 was analyzed in vitro and in vivo. The results revealed that pHsp33 was successfully expressed both in transfected hirame natural embryo cell lines and injected flounder muscle, suggesting the functionality of pHsp33 to express Hsp33 protein. Fish, when intramuscularly injected with pHsp33 vaccine, exhibited the production of specific antibodies, upregulation of immune related genes expression in the head kidney and increase of sIgM+, CD4-1+ and CD4-2+ lymphocytes in peripheral blood, spleen and head kidney, which indicated the activation of humoral and cellular immune responses. Moreover, pHsp33 upregulated the expression of immune related genes at the inoculation site, indicating the activation of local immune response. In addition, pHsp33 vaccinated flounder provided a relative percent survival of 42.86% and inhibited the pathological lesion in liver following V. anguillarum challenge. In general, the results revealed that pHsp33 could elicit local and system immune responses and confer protection for flounder, suggesting pHsp33 could serve as a DNA vaccine candidate for the control of V. anguillarum infection.

CnoX Is a Chaperedoxin: A Holdase that Protects Its Substrates from Irreversible Oxidation.

Bleach (HOCl) is a powerful oxidant that kills bacteria in part by causing protein aggregation. It inactivates ATP-dependent chaperones, rendering cellular proteins mostly dependent on holdases. Here we identified Escherichia coli CnoX (YbbN) as a folding factor that, when activated by bleach via chlorination, functions as an efficient holdase, protecting the substrates of the major folding systems GroEL/ES and DnaK/J/GrpE. Remarkably, CnoX uniquely combines this function with the ability to prevent the irreversible oxidation of its substrates. This dual activity makes CnoX the founding member of a family of proteins, the "chaperedoxins." Because CnoX displays a thioredoxin fold and a tetratricopeptide (TPR) domain, two structural motifs conserved in all organisms, this investigation sets the stage for the discovery of additional chaperedoxins in bacteria and eukaryotes that could cooperate with proteins from both the Hsp60 and Hsp70 families.

A Role of Metastable Regions and Their Connectivity in the Inactivation of a Redox-Regulated Chaperone and Its Inter-Chaperone Crosstalk.

AIMS: A recently discovered group of conditionally disordered chaperones share a very unique feature; they need to lose structure to become active as chaperones. This activation mechanism makes these chaperones particularly suited to respond to protein-unfolding stress conditions, such as oxidative unfolding. However, the role of this disorder in stress-related activation, chaperone function, and the crosstalk with other chaperone systems is not yet clear. Here, we focus on one of the members of the conditionally disordered chaperones, a thiol-redox switch of the bacterial proteostasis system, Hsp33. RESULTS: By modifying the Hsp33's sequence, we reveal that the metastable region has evolved to abolish redox-dependent chaperone activity, rather than enhance binding affinity for client proteins. The intrinsically disordered region of Hsp33 serves as an anchor for the reduced, inactive state of Hsp33, and it dramatically affects the crosstalk with the synergetic chaperone system, DnaK/J. Using mass spectrometry, we describe the role that the metastable region plays in determining client specificity during normal and oxidative stress conditions in the cell. Innovation and Conclusion: We uncover a new role of protein plasticity in Hsp33's inactivation, client specificity, crosstalk with the synergistic chaperone system DnaK/J, and oxidative stress-specific interactions in bacteria. Our results also suggest that Hsp33 might serve as a member of the house-keeping proteostasis machinery, tasked with maintaining a "healthy" proteome during normal conditions, and that this function does not depend on the metastable linker region. Antioxid. Redox Signal. 27, 1252-1267.

HSP33 in eukaryotes - an evolutionary tale of a chaperone adapted to photosynthetic organisms.

HSP33 was originally identified in bacteria as a redox-sensitive chaperone that protects unfolded proteins from aggregation. Here, we describe a eukaryote ortholog of HSP33 from the green algae Chlamydomonas reinhardtii, which appears to play a protective role under light-induced oxidizing conditions. The algal HSP33 exhibits chaperone activity, as shown by citrate synthase aggregation assays. Studies from the Jakob laboratory established that activation of the bacterial HSP33 upon its oxidation initiates by the release of pre-bound Zn from the well conserved Zn-binding motif Cys-X-Cys-Xn -Cys-X-X-Cys, and is followed by significant structural changes (Reichmann et al., ). Unlike the bacterial protein, the HSP33 from C. reinhardtii had lost the first cysteine residue of its center, diminishing Zn-binding activity under all conditions. As a result, the algal protein can be easily activated by minor structural changes in response to oxidation and/or excess heat. An attempt to restore the missing first cysteine did not have a major effect on Zn-binding and on the mode of activation. Replacement of all remaining cysteines abolished completely any residual Zn binding, although the chaperone activation was maintained. A phylogenetic analysis of the algal HSP33 showed that it clusters with the cyanobacterial protein, in line with its biochemical localization to the chloroplast. Indeed, expression of the algal HSP33 increases in response to light-induced oxidative stress, which is experienced routinely by photosynthetic organisms. Despite the fact that no ortholog could be found in higher eukaryotes, its abundance in all algal species examined could have a biotechnological relevance.

Type I cell ROS kinetics under hypoxia in the intact mouse carotid body ex vivo: a FRET-based study.

Reactive oxygen species (ROS) mainly originating from NADPH oxidases have been shown to be involved in the carotid body (CB) oxygen-sensing cascade. For measuring ROS kinetics, type I cells of the mouse CB in an ex vivo preparation were transfected with the ROS sensor construct FRET-HSP33. After 2 days of tissue culture, type I cells expressed FRET-HSP33 as shown by immunohistochemistry. In one population of CBs, 5 min of hypoxia induced a significant and reversible decrease of type I cell ROS levels (n = 9 CBs; P < 0.015), which could be inhibited by 4-(2-aminoethyl)benzensulfonylfluorid (AEBSF), a highly specific inhibitor of the NADPH oxidase subunits p47(phox) and p67(phox). In another population of CBs, however, 5 min of hypoxia induced a significant and reversible increase of ROS levels in type I cells (n = 8 CBs; P < 0.05), which was slightly enhanced by administration of 3 mM AEBSF. These different ROS kinetics seemed to coincide with different mice breeding conditions. Type I cells of both populations showed a typical hypoxia-induced membrane potential (MP) depolarization, which could be inhibited by 3 mM AEBSF. ROS and MP closely followed the hypoxic decrease in CB tissue oxygen as measured with an O2-sensitive dye. We conclude that attenuated p47(phox) subunit activity of the NADPH oxidase under hypoxia is the physiological trigger for type I cell MP depolarization probably due to ROS decrease, whereas the observed ROS increase has no influence on type I cell MP kinetics under hypoxia.