Redox-regulated chaperones in cell stress responses.

Proteostasis and redox homeostasis are tightly interconnected and most protein quality control pathways are under direct redox regulation which allow cells to immediately respond to oxidative stress conditions. The activation of ATP-independent chaperones serves as a first line of defense to counteract oxidative unfolding and aggregation of proteins. Conserved cysteine residues evolved as redox-sensitive switches which upon reversible oxidation induce substantial conformational rearrangements and the formation of chaperone-active complexes. In addition to harnessing unfolding proteins, these chaperone holdases interact with ATP-dependent chaperone systems to facilitate client refolding and restoring proteostasis during stress recovery. This minireview gives an insight into highly orchestrated mechanisms regulating the stress-specific activation and inactivation of redox-regulated chaperones and their role in cell stress responses.

Escherichia coli RclA is a highly active hypothiocyanite reductase.

Hypothiocyanite and hypothiocyanous acid (OSCN-/HOSCN) are pseudohypohalous acids released by the innate immune system which are capable of rapidly oxidizing sulfur-containing amino acids, causing significant protein aggregation and damage to invading bacteria. HOSCN is abundant in saliva and airway secretions and has long been considered a highly specific antimicrobial that is nearly harmless to mammalian cells. However, certain bacteria, commensal and pathogenic, are able to escape damage by HOSCN and other harmful antimicrobials during inflammation, which allows them to continue to grow and, in some cases, cause severe disease. The exact genes or mechanisms by which bacteria respond to HOSCN have not yet been elucidated. We have found, in Escherichia coli, that the flavoprotein RclA, previously implicated in reactive chlorine resistance, reduces HOSCN to thiocyanate with near-perfect catalytic efficiency and strongly protects E. coli against HOSCN toxicity. This is notable in E. coli because this species thrives in the chronically inflamed environment found in patients with inflammatory bowel disease and is able to compete with and outgrow other important commensal organisms, suggesting that HOSCN may be a relevant antimicrobial in the gut, which has not previously been explored. RclA is conserved in a variety of epithelium-colonizing bacteria, implicating its HOSCN reductase activity in a variety of host-microbe interactions. We show that an rclA mutant of the probiotic Limosilactobacillus reuteri is sensitive to HOSCN and that RclA homologs from Staphylococcus aureus, Streptococcus pneumoniae, and Bacteroides thetaiotaomicron all have potent protective activity against HOSCN when expressed in E. coli.

From guide to guard-activation mechanism of the stress-sensing chaperone Get3.

Oxidative stress conditions can cause ATP depletion, oxidative protein unfolding, and potentially toxic protein aggregation. To alleviate this proteotoxic stress, the highly conserved yeast protein, Get3, switches from its guiding function as an ATP-dependent targeting factor for tail-anchored proteins to its guarding function as an ATP-independent molecular chaperone that prevents irreversible protein aggregation. Here, we demonstrate that activation of Get3's chaperone function follows a tightly orchestrated multi-step process, centered around the redox status of two conserved cysteines, whose reactivity is directly controlled by Get3's nucleotide-binding state. Thiol oxidation causes local unfolding and the transition into chaperone-active oligomers. Vice versa, inactivation requires the reduction of Get3's cysteines followed by ATP-binding, which allows the transfer of bound client proteins to ATP-dependent chaperone systems for their effective refolding. Manipulating this fine-tuned cycle of activation and inactivation in yeast impairs oxidative stress resistance and growth, illustrating the necessity to tightly control Get3's intrinsic chaperone function.

Thiol-based switching mechanisms of stress-sensing chaperones.

Thiol-based redox switches evolved as efficient post-translational regulatory mechanisms that enable individual proteins to rapidly respond to sudden environmental changes. While some protein functions need to be switched off to save resources and avoid potentially error-prone processes, protective functions become essential and need to be switched on. In this review, we focus on thiol-based activation mechanisms of stress-sensing chaperones. Upon stress exposure, these chaperones convert into high affinity binding platforms for unfolding proteins and protect cells against the accumulation of potentially toxic protein aggregates. Their chaperone activity is independent of ATP, a feature that becomes especially important under oxidative stress conditions, where cellular ATP levels drop and canonical ATP-dependent chaperones no longer operate. Vice versa, reductive inactivation and substrate release require the restoration of ATP levels, which ensures refolding of client proteins by ATP-dependent foldases. We will give an overview over the different strategies that cells evolved to rapidly increase the pool of ATP-independent chaperones upon oxidative stress and provide mechanistic insights into how stress conditions are used to convert abundant cellular proteins into ATP-independent holding chaperones.

Mycobacterium tuberculosis Rv0991c Is a Redox-Regulated Molecular Chaperone.

The bacterial pathogen Mycobacterium tuberculosis is the leading cause of death by an infectious disease among humans. Here, we describe a previously uncharacterized M. tuberculosis protein, Rv0991c, as a molecular chaperone that is activated by oxidation. Rv0991c has homologs in most bacterial lineages and appears to function analogously to the well-characterized Escherichia coli redox-regulated chaperone Hsp33, despite a dissimilar protein sequence. Rv0991c is transcriptionally coregulated with hsp60 and hsp70 chaperone genes in M. tuberculosis, suggesting that Rv0991c functions with these chaperones in maintaining protein quality control. Supporting this hypothesis, we found that, like oxidized Hsp33, oxidized Rv0991c prevents the aggregation of a model unfolded protein in vitro and promotes its refolding by the M. tuberculosis Hsp70 chaperone system. Furthermore, Rv0991c interacts with DnaK and can associate with many other M. tuberculosis proteins. We therefore propose that Rv0991c, which we named "Ruc" (redox-regulated protein with unstructured C terminus), represents a founding member of a new chaperone family that protects M. tuberculosis and other species from proteotoxicity during oxidative stress.IMPORTANCEM. tuberculosis infections are responsible for more than 1 million deaths per year. Developing effective strategies to combat this disease requires a greater understanding of M. tuberculosis biology. As in all cells, protein quality control is essential for the viability of M. tuberculosis, which likely faces proteotoxic stress within a host. Here, we identify an M. tuberculosis protein, Ruc, that gains chaperone activity upon oxidation. Ruc represents a previously unrecognized family of redox-regulated chaperones found throughout the bacterial superkingdom. Additionally, we found that oxidized Ruc promotes the protein-folding activity of the essential M. tuberculosis Hsp70 chaperone system. This work contributes to a growing body of evidence that oxidative stress provides a particular strain on cellular protein stability.

Developmental ROS individualizes organismal stress resistance and lifespan.

A central aspect of aging research concerns the question of when individuality in lifespan arises1. Here we show that a transient increase in reactive oxygen species (ROS), which occurs naturally during early development in a subpopulation of synchronized Caenorhabditis elegans, sets processes in motion that increase stress resistance, improve redox homeostasis and ultimately prolong lifespan in those animals. We find that these effects are linked to the global ROS-mediated decrease in developmental histone H3K4me3 levels. Studies in HeLa cells confirmed that global H3K4me3 levels are ROS-sensitive and that depletion of H3K4me3 levels increases stress resistance in mammalian cell cultures. In vitro studies identified SET1/MLL histone methyltransferases as redox sensitive units of the H3K4-trimethylating complex of proteins (COMPASS). Our findings implicate a link between early-life events, ROS-sensitive epigenetic marks, stress resistance and lifespan.

An essential thioredoxin-type protein of Trypanosoma brucei acts as redox-regulated mitochondrial chaperone.

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes.

The role of thiols in antioxidant systems.

The sulfur biochemistry of the thiol group endows cysteines with a number of highly specialized and unique features that enable them to serve a variety of different functions in the cell. Typically highly conserved in proteins, cysteines are predominantly found in functionally or structurally crucial regions, where they act as stabilizing, catalytic, metal-binding and/or redox-regulatory entities. As highly abundant low molecular weight thiols, cysteine thiols and their oxidized disulfide counterparts are carefully balanced to maintain redox homeostasis in various cellular compartments, protect organisms from oxidative and xenobiotic stressors and partake actively in redox-regulatory and signaling processes. In this review, we will discuss the role of protein thiols as scavengers of hydrogen peroxide in antioxidant enzymes, use thiol peroxidases to exemplify how protein thiols contribute to redox signaling, provide an overview over the diverse set of low molecular weight thiol-based redox systems found in biology, and illustrate how thiol-based redox systems have evolved not only to protect against but to take full advantage of a world full of molecular oxygen.

A glutaredoxin in the mitochondrial intermembrane space has stage-specific functions in the thermo-tolerance and proliferation of African trypanosomes.

Trypanosoma brucei glutaredoxin 2 (Grx2) is a dithiol glutaredoxin that is specifically located in the mitochondrial intermembrane space. Bloodstream form parasites lacking Grx2 or both, Grx2 and the cytosolic Grx1, are viable in vitro and infectious to mice suggesting that neither oxidoreductase is needed for survival or infectivity to mammals. A 37 °C to 39 °C shift changes the cellular redox milieu of bloodstream cells to more oxidizing conditions and induces a significantly stronger growth arrest in wildtype parasites compared to the mutant cells. Grx2-deficient cells ectopically expressing the wildtype form of Grx2 with its C31QFC34 active site, but not the C34S mutant, regain the sensitivity of the parental strain, indicating that the physiological role of Grx2 requires both active site cysteines. In the procyclic insect stage of the parasite, Grx2 is essential. Both alleles can be replaced if procyclic cells ectopically express authentic or C34S, but not C31S/C34S Grx2, pointing to a redox role that relies on a monothiol mechanism. RNA-interference against Grx2 causes a virtually irreversible proliferation defect. The cells adopt an elongated morphology but do not show any significant alteration in the cell cycle. The growth retardation is attenuated by high glucose concentrations. Under these conditions, procyclic cells obtain ATP by substrate level phosphorylation suggesting that Grx2 might regulate a respiratory chain component.

Development of Novel Peptide-Based Michael Acceptors Targeting Rhodesain and Falcipain-2 for the Treatment of Neglected Tropical Diseases (NTDs).

This paper describes the development of a class of peptide-based inhibitors as novel antitrypanosomal and antimalarial agents. The inhibitors are based on a characteristic peptide sequence for the inhibition of the cysteine proteases rhodesain of Trypanosoma brucei rhodesiense and falcipain-2 of Plasmodium falciparum. We exploited the reactivity of novel unsaturated electrophilic functions such as vinyl-sulfones, -ketones, -esters, and -nitriles. The Michael acceptors inhibited both rhodesain and falcipain-2, at nanomolar and micromolar levels, respectively. In particular, the vinyl ketone 3b has emerged as a potent rhodesain inhibitor (k2nd = 67 × 106 M-1 min-1), endowed with a picomolar binding affinity (Ki = 38 pM), coupled with a single-digit micromolar activity against Trypanosoma brucei brucei (EC50 = 2.97 µM), thus being considered as a novel lead compound for the discovery of novel effective antitrypanosomal agents.

Stress-Induced Protein S-Glutathionylation and S-Trypanothionylation in African Trypanosomes-A Quantitative Redox Proteome and Thiol Analysis.

AIMS: Trypanosomatids have a unique trypanothione-based thiol redox metabolism. The parasite-specific dithiol is synthesized from glutathione and spermidine, with glutathionylspermidine as intermediate catalyzed by trypanothione synthetase. In this study, we address the oxidative stress response of African trypanosomes with special focus on putative protein S-thiolation. RESULTS: Challenging bloodstream Trypanosoma brucei with diamide, H2O2 or hypochlorite results in distinct levels of reversible overall protein S-thiolation. Quantitative proteome analyses reveal 84 proteins oxidized in diamide-stressed parasites. Fourteen of them, including several essential thiol redox proteins and chaperones, are also enriched when glutathione/glutaredoxin serves as a reducing system indicating S-thiolation. In parasites exposed to H2O2, other sets of proteins are modified. Only three proteins are S-thiolated under all stress conditions studied in accordance with a highly specific response. H2O2 causes primarily the formation of free disulfides. In contrast, in diamide-treated cells, glutathione, glutathionylspermidine, and trypanothione are almost completely protein bound. Remarkably, the total level of trypanothione is decreased, whereas those of glutathione and glutathionylspermidine are increased, indicating partial hydrolysis of protein-bound trypanothione. Depletion of trypanothione synthetase exclusively induces protein S-glutathionylation. Total mass analyses of a recombinant peroxidase treated with T(SH)2 and either diamide or hydrogen peroxide verify protein S-trypanothionylation as stable modification. INNOVATION: Our data reveal for the first time that trypanosomes employ protein S-thiolation when exposed to exogenous and endogenous oxidative stresses and trypanothione, despite its dithiol character, forms protein-mixed disulfides. CONCLUSION: The stress-specific responses shown here emphasize protein S-trypanothionylation and S-glutathionylation as reversible protection mechanism in these parasites. Antioxid. Redox Signal. 27, 517-533.

Is there a need for ophthalmological examinations after a first seizure in paediatric patients?

In contrast to the recommendations of the International Liga against Epilepsy, many hospitals perform routinely complete ophthalmological examinations in children admitted after a first seizure. As there is no study available to date to prove the benefit of complete eye examinations in first seizure diagnosis, we conducted a study to analyse the value of a complete ophthalmological examination. All children aged 1 month to 18 years who were admitted to the children's university hospital of Leipzig with the clinical diagnosis of a first convulsive or non-convulsive afebrile seizure between 1999 and August 2005 were investigated. All children who had obtained a complete ophthalmological examination within 72 h after the seizure were included in the observational study. A total of 310 children were analysed in the study. Two hundred thirty patients had a tonic-clonic afebrile seizure, the others focal, complex-partial seizures or absences. Two hundred seven out of 310 children showed no ophthalmological pathologies. Eighty-three children had refraction anomalies or strabism, 18 children had optic atrophy, three had congenital eye muscle paresis, and three had malformations. A 16-year-old girl had a homonymous quadrantanopia due to an occipital glioglioma that caused the seizure. An 11-year-old girl had a retinal haemorrhage without any brain lesions after a fall caused by a first tonic-clonic seizure. None of the ophthalmological findings influenced directly the immediate clinical course of diagnosis and treatment of the seizure. Our data suggest that routine ophthalmological examination in all children does not have additional benefit in the first seizure diagnosis management.

Effectiveness of an intensive multidisciplinary headache treatment program.

OBJECTIVE: To investigate if the effectiveness of a 96-hour multidisciplinary headache treatment program exceeds the effectiveness of a 20-hour program and primary care. BACKGROUND: When dealing with chronic back pain, low-intensity multidisciplinary treatment yields no significantly better results than standard care and monodisciplinary therapy; however, high-intensity treatment does. For multidisciplinary headache treatment, such comparisons are not yet available. In a previous study undertaken by our Pain Center, the outcome of a minimal multidisciplinary intervention model (20-hour) did not exceed primary care. METHODS: Forty-two patients suffering from frequent headaches (20 +/- 9 headache days/month; range: 8-30) were treated and evaluated in a 96-hour group program. The results were compared with the outcomes of the previous study. Subjects who had undergone either the 20-hour multidisciplinary program or the primary care were used as historical control groups. FINDINGS: A significant reduction in migraine days (P < .001), tension-type headache days (P < .001), frequency of migraine attacks (P = .004), and depression score (P < .001) was seen at the follow-up after 22 (+/-2) weeks. Comparing the intensive multidisciplinary program with primary care, repeated measures ANOVAs revealed significant time x group interactions for migraine days (P = .020), tension-type headache days (P = .016), and frequency of migraine attacks (P = .016). In comparison with the 20-hour multidisciplinary program, the 96-hour program showed significantly better effects only in the reduction of migraine days (P = .037) and depression score (P = .003). The responder-rates (> or =50% improvement) in the 96-hour program were significantly higher than in the 20-hour program (migraine days, P = .008; tension-type headache days, P = .044) and primary care (migraine days, P = .007; tension-type headache days, P = .003; tension-type headache intensity, P = .037). The effect sizes were small to medium in the 96-hour program. Particularly with the reduction of migraine symptomatology, the 96-hour program performed better than the 20-hour program, which produced only negligible or small effects. CONCLUSIONS: Intensive multidisciplinary headache treatment is highly effective for patients with chronic headaches. Furthermore, migraine symptomatology responds especially well to this intensive treatment program, whereas effects on tension-type headaches were realized by both multidisciplinary programs. Randomized controlled trials and subgroup analysis are needed to find out if these results can be replicated and which patient characteristics allow for sufficient improvements for headache sufferers even with less complex treatment.