Csaba Sőti

A Role for SKN-1/Nrf in Pathogen Resistance and Immunosenescence in Caenorhabditis elegans

A proper immune response ensures survival in a hostile environment and promotes longevity. Recent evidence indicates that innate immunity, beyond antimicrobial effectors, also relies on host-defensive mechanisms. The Caenorhabditis elegans transcription factor SKN-1 regulates xenobiotic and oxidative stress responses and contributes to longevity, however, its role in immune defense is unknown. Here we show that SKN-1 is required for C. elegans pathogen resistance against both Gram-negative Pseudomonas aeruginosa and Gram-positive Enterococcus faecalis bacteria. Exposure to P. aeruginosa leads to SKN-1 accumulation in intestinal nuclei and transcriptional activation of two SKN-1 target genes, gcs-1 and gst-4. Both the Toll/IL-1 Receptor domain protein TIR-1 and the p38 MAPK PMK-1 are required for SKN-1 activation by PA14 exposure. We demonstrate an early onset of immunosenescence with a concomitant age-dependent decline in SKN-1-dependent target gene activation, and a requirement of SKN-1 to enhance pathogen resistance in response to longevity-promoting interventions, such as reduced insulin/IGF-like signaling and preconditioning H2O2 treatment. Finally, we find that wdr-23(RNAi)-mediated constitutive SKN-1 activation results in excessive transcription of target genes, confers oxidative stress tolerance, but impairs pathogen resistance. Our findings identify SKN-1 as a novel regulator of innate immunity, suggests its involvement in immunosenescence and provide an important crosstalk between pathogenic stress signaling and the xenobiotic/oxidative stress response.

Protein stress and stress proteins: Implications in aging and disease

Environmantal stress induces damage that activates an adaptive response in any organism. The cellular stress response is based on the induction of cytoprotective proteins, the so called stress or heat shock proteins. The stress response as well as stress proteins are ubiquitous, highly conserved mechanism, and genes, respectively, already present in prokaryotes. Chaperones protect the proteome against conformational damage, promoting the function of protein networks. Protein damage takes place during aging and in several degenerative diseases, and presents a threat to overload the cellular defense mechanisms. The preservation of a robust stress response and protein disposal is indispensable for health and longevity. This review summarizes the present knowledge of protein damage, turnover, and the stress response in aging and degenerative diseases.

Protein homeostasis and molecular chaperones in aging

Molecular chaperones are ubiquitous, highly conserved proteins responsible for the maintenance of protein folding homeostasis in cells. Environmental stress causes proteotoxic damage, which triggers chaperone induction and the subsequent reparation of cellular damage by chaperones, including disposing irreparable protein ensembles. We summarize the current view of protein damage, turnover, the stress response and chaperone function in aging, and review novel data showing that accumulation of misfolded proteins outcompete and overload the limited resources of the protein folding, maintenance and turnover system, compromising general protein homeastasis and cellular function. Possible involvement of chaperones and proteolysis in immunosenescence is highlighted. Defects in zinc metabolism are also addressed in relation to aging and changes in chaperone levels.

Ageing as a price of cooperation and complexity

The network concept is increasingly used for the description of complex systems. Here, we summarize key aspects of the evolvability and robustness of the hierarchical network set of macromolecules, cells, organisms and ecosystems. Listing the costs and benefits of cooperation as a necessary behaviour to build this network hierarchy, we outline the major hypothesis of the paper: the emergence of hierarchical complexity needs cooperation leading to the ageing (i.e. gradual deterioration) of the constituent networks. A stable environment develops cooperation leading to over‐optimization, and forming an ‘always‐old’ network, which accumulates damage, and dies in an apoptosis‐like process. A rapidly changing environment develops competition forming a ‘forever‐young’ network, which may suffer an occasional over‐perturbation exhausting system resources, and causing death in a necrosis‐like process. Giving a number of examples we demonstrate how cooperation evokes the gradual accumulation of damage typical to ageing. Finally, we show how various forms of cooperation and consequent ageing emerge as key elements in all major steps of evolution from the formation of protocells to the establishment of the globalized, modern human society.

Hsp90 chaperones PPARγ and regulates differentiation and survival of 3T3-L1 adipocytes

Adipose tissue dysregulation has a major role in various human diseases. The peroxisome proliferator-activated receptor-γ (PPARγ) is a key regulator of adipocyte differentiation and function, as well as a target of insulin-sensitizing drugs. The Hsp90 chaperone stabilizes a diverse set of signaling ‘client’ proteins, thereby regulates various biological processes. Here we report a novel role for Hsp90 in controlling PPARγ stability and cellular differentiation. Specifically, we show that the Hsp90 inhibitors geldanamycin and novobiocin efficiently impede the differentiation of murine 3T3-L1 preadipocytes. Geldanamycin at higher concentrations also inhibits the survival of both developing and mature adipocytes, respectively. Further, Hsp90 inhibition disrupts an Hsp90-PPARγ complex, leads to the destabilization and proteasomal degradation of PPARγ, and inhibits the expression of PPARγ target genes, identifying PPARγ as an Hsp90 client. A similar destabilization of PPARγ and a halt of adipogenesis also occur in response to protein denaturing stresses caused by a single transient heat-shock or proteasome inhibition. Recovery from stress restores PPARγ stability and adipocyte differentiation. Thus, our findings reveal Hsp90 as a critical stress-responsive regulator of adipocyte biology and offer a potential therapeutic target in obesity and the metabolic syndrome.

Misfolded proteins inhibit proliferation and promote stress-induced death in SV40-transformed mammalian cells

Protein misfolding is implicated in neurodegenerative diseases and occurs in aging. However, the contribution of the misfolded ensembles to toxicity remains largely unknown. Here we introduce 2 primate cell models of destabilized proteins devoid of specific cellular functions and interactors, as bona fide mis‐folded proteins, allowing us to isolate the gain‐of‐function of non‐native structures. Both GFP‐degron and a mutant chloramphenicol‐acetyltransferase fused to GFP (GFP‐Δ9CAT) form perinuclear aggregates, are degraded by the proteasome, and colocalize with and induce the chaperone Hsp70 (HSPA1A/B) in COS‐7 cells. We find that misfolded proteins neither significantly compromise chaperone‐mediated folding capacity nor induce cell death. However, they do induce growth arrest in cells that are unable to degrade them and promote stress‐induced death upon proteasome inhibition by MG‐132 and heat shock. Finally, we show that overexpression of all heat‐shock factor‐1 (HSF1) and Hsp70 proteins, as well as wild‐type and deacetylase‐deficient (H363Y) SIRT1, rescue survival upon stress, implying a noncatalytic action of SIRT1 in response to protein misfolding. Our study establishes a novel model and extends our knowledge on the mechanism of the function‐independent proteotoxicity of misfolded proteins in dividing cells.—Arslan, M. A., Chikina, M., Csermely, P., Sőti, C. Misfolded proteins inhibit proliferation and promote stress‐induced death in SV40‐transformed mammalian cells. FASEB J. 26, 766–777 (2012). www.fasebj.org

RNA interference links oxidative stress to the inhibition of heat stress adaptation.

UNLABELLED Increased oxidative stress is associated with various diseases and aging, while adaptation to heat stress is an important determinant of survival and contributes to longevity. However, the impact of oxidative stress on heat resistance remains largely unclear. AIM In this study we investigated how oxidative stress impinges on heat stress responses. RESULTS We report that hydrogen-peroxide (H(2)O(2)) pretreatment inhibits both acquired thermotolerance and heat-induced Hsp70 expression in mammalian cells, as well as acquired thermotolerance in the nematode Caenorhabditis elegans, via RNA interference. Moreover, we demonstrate that elimination of RNA interference by silencing key enzymes in microRNA biogenesis, dcr-1 or pash-1, restores the diminished intrinsic thermotolerance of aged and H(2)O(2)-elimination compromised (catalase-2 and peroxiredoxin-2 deficient) worms. INNOVATION AND CONCLUSION These results uncover a novel post-transcriptional element in the regulation of heat stress adaptation under oxidative conditions that may have implications in disease susceptibility and aging.

Characterization of the nucleotide binding properties of the 90 kDa heat shock protein (Hsp90)

The recent crystallization and structural analysis of the ATP(ADP)-complex of the N-terminal domain of the 90 kDa heat shock protein (Hsp90) confirmed our earlier findings on the ATP-binding properties of Hsp90. Here we further characterize the nucleotide binding of Hsp90 by demonstrating that surface plasmon resonance measurements also indicate a low-affinity binding of ATP to Hsp90 and that [α-32P]ATP seems to have an equal preference for monomers, dimers and oligomers of Hsp90 on native polyacrylamide gels. Finally we discuss some of our results which raise the possibility that Hsp90 has two nucleotide binding sites (one in its N-terminal and another in the C-terminal domain) and that the nucleotide binding to Hsp90 dimers may display a positive cooperativity under some special conditions. The submillimolar binding affinity of ATP to Hsp90 allows the regulation of some Hsp90-related functions just in the range of ATP-level fluctuations during stress or during the cell cycle.

LPS-Induced Delayed Preconditioning Is Mediated by Hsp90 and Involves the Heat Shock Response in Mouse Kidney

Introduction We and others demonstrated previously that preconditioning with endotoxin (LPS) protected from a subsequent lethal LPS challenge or from renal ischemia-reperfusion injury (IRI). LPS is effective in evoking the heat shock response, an ancient and essential cellular defense mechanism, which plays a role in resistance to, and recovery from diseases. Here, by using the pharmacological Hsp90 inhibitor novobiocin (NB), we investigated the role of Hsp90 and the heat shock response in LPS-induced delayed renal preconditioning. Methods Male C57BL/6 mice were treated with preconditioning (P: 2 mg/kg, ip.) and subsequent lethal (L: 10 mg/kg, ip.) doses of LPS alone or in combination with NB (100 mg/kg, ip.). Controls received saline (C) or NB. Results Preconditioning LPS conferred protection from a subsequent lethal LPS treatment. Importantly, the protective effect of LPS preconditioning was completely abolished by a concomitant treatment with NB. LPS induced a marked heat shock protein increase as demonstrated by Western blots of Hsp70 and Hsp90. NB alone also stimulated Hsp70 and Hsp90 mRNA but not protein expression. However, Hsp70 and Hsp90 protein induction in LPS-treated mice was abolished by a concomitant NB treatment, demonstrating a NB-induced impairment of the heat shock response to LPS preconditioning. Conclusion LPS-induced heat shock protein induction and tolerance to a subsequent lethal LPS treatment was prevented by the Hsp90 inhibitor, novobiocin. Our findings demonstrate a critical role of Hsp90 in LPS signaling, and a potential involvement of the heat shock response in LPS-induced preconditioning.

Distinct roles of the RasGAP family proteins in C. elegans associative learning and memory

The Ras GTPase activating proteins (RasGAPs) are regulators of the conserved Ras/MAPK pathway. Various roles of some of the RasGAPs in learning and memory have been reported in different model systems, yet, there is no comprehensive study to characterize all gap genes in any organism. Here, using reverse genetics and neurobehavioural tests, we studied the role of all known genes of the rasgap family in C. elegans in associative learning and memory. We demonstrated that their proteins are implicated in different parts of the learning and memory processes. We show that gap-1 contribute redundantly with gap-3 to the chemosensation of volatile compounds, gap-1 plays a major role in associative learning, while gap-2 and gap-3 are predominantly required for short- and long-term associative memory. Our results also suggest that the C. elegans Ras orthologue let-60 is involved in multiple processes during learning and memory. Thus, we show that the different classes of RasGAP proteins are all involved in cognitive function and their complex interplay ensures the proper formation and storage of novel information in C. elegans.