Antifungal drug resistance evoked via RNAi-dependent epimutations.

Microorganisms evolve via a range of mechanisms that may include or involve sexual/parasexual reproduction, mutators, aneuploidy, Hsp90 and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show that the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidylprolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the fkbA gene, giving rise to drug-resistant epimutants. FK506-resistant epimutants readily reverted to the drug-sensitive wild-type phenotype when grown without exposure to the drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNAs and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves the generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as a template to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.

Phosphate is the third nutrient monitored by TOR in Candida albicans and provides a target for fungal-specific indirect TOR inhibition.

The Target of Rapamycin (TOR) pathway regulates morphogenesis and responses to host cells in the fungal pathogen Candida albicans Eukaryotic Target of Rapamycin complex 1 (TORC1) induces growth and proliferation in response to nitrogen and carbon source availability. Our unbiased genetic approach seeking unknown components of TORC1 signaling in C. albicans revealed that the phosphate transporter Pho84 is required for normal TORC1 activity. We found that mutants in PHO84 are hypersensitive to rapamycin and in response to phosphate feeding, generate less phosphorylated ribosomal protein S6 (P-S6) than the WT. The small GTPase Gtr1, a component of the TORC1-activating EGO complex, links Pho84 to TORC1. Mutants in Gtr1 but not in another TORC1-activating GTPase, Rhb1, are defective in the P-S6 response to phosphate. Overexpression of Gtr1 and a constitutively active Gtr1Q67L mutant suppresses TORC1-related defects. In Saccharomyces cerevisiae pho84 mutants, constitutively active Gtr1 suppresses a TORC1 signaling defect but does not rescue rapamycin hypersensitivity. Hence, connections from phosphate homeostasis (PHO) to TORC1 may differ between C. albicans and S. cerevisiae The converse direction of signaling from TORC1 to the PHO regulon previously observed in S. cerevisiae was genetically shown in C. albicans using conditional TOR1 alleles. A small molecule inhibitor of Pho84, a Food and Drug Administration-approved drug, inhibits TORC1 signaling and potentiates the activity of the antifungals amphotericin B and micafungin. Anabolic TORC1-dependent processes require significant amounts of phosphate. Our study shows that phosphate availability is monitored and also controlled by TORC1 and that TORC1 can be indirectly targeted by inhibiting Pho84.

A non-canonical RNA degradation pathway suppresses RNAi-dependent epimutations in the human fungal pathogen Mucor circinelloides.

Mucorales are a group of basal fungi that includes the casual agents of the human emerging disease mucormycosis. Recent studies revealed that these pathogens activate an RNAi-based pathway to rapidly generate drug-resistant epimutant strains when exposed to stressful compounds such as the antifungal drug FK506. To elucidate the molecular mechanism of this epimutation pathway, we performed a genetic analysis in Mucor circinelloides that revealed an inhibitory role for the non-canonical RdRP-dependent Dicer-independent silencing pathway, which is an RNAi-based mechanism involved in mRNA degradation that was recently identified. Thus, mutations that specifically block the mRNA degradation pathway, such as those in the genes r3b2 and rdrp3, enhance the production of drug resistant epimutants, similar to the phenotype previously described for mutation of the gene rdrp1. Our genetic analysis also revealed two new specific components of the epimutation pathway related to the quelling induced protein (qip) and a Sad-3-like helicase (rnhA), as mutations in these genes prevented formation of drug-resistant epimutants. Remarkably, drug-resistant epimutant production was notably increased in M. circinelloides f. circinelloides isolates from humans or other animal hosts. The host-pathogen interaction could be a stressful environment in which the phenotypic plasticity provided by the epimutant pathway might provide an advantage for these strains. These results evoke a model whereby balanced regulation of two different RNAi pathways is determined by the activation of the RNAi-dependent epimutant pathway under stress conditions, or its repression when the regular maintenance of the mRNA degradation pathway operates under non-stress conditions.

Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen Cryptococcus neoformans.

Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that loss of Crz1 confers phenotypes intermediate between wild-type and calcineurin mutants, and demonstrate that deletion of the calcineurin docking domain results in the inability of Crz1 to translocate into the nucleus under thermal stress. RNA-sequencing revealed 102 genes that are regulated in a calcineurin-Crz1-dependent manner at 37°C. The majority of genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin-Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. We identified three calcineurin-dependent response element motifs within the promoter regions of calcineurin-Crz1 target genes, and show that Crz1 binding to target gene promoters is increased upon thermal stress in a calcineurin-dependent fashion. Additionally, we found a large set of genes independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output in C. neoformans appears to be adapted to promote fungal virulence.

The use of cobalt chloride as a chemical hypoxia model.

The use of hypoxia models in cell culture has allowed the characterization of the hypoxia response at the cellular, biochemical and molecular levels. Although a decrease in oxygen concentration is the optimal hypoxia model, the problem faced by many researchers is access to a hypoxia chamber or a CO2 incubator with regulated oxygen levels, which is not possible in many laboratories. Several alternative models have been used to mimic hypoxia. One of the most commonly used models is cobalt chloride-induced chemical hypoxia because it stabilizes hypoxia inducible factors 1α and 2α under normoxic conditions. This model has several advantages, and currently, there is a substantial amount of scattered information about how this model works. This review describes the characteristics of the model, as well as the biochemical and molecular bases that support it. The regulation of hypoxia inducible factors by oxygen and the role of CoCl2 are explained to understand the most accepted bases of the CoCl2 -induced hypoxia model. The different current hypotheses that explain the establishment of hypoxic conditions using CoCl2 are also described. Finally, based on the different observations reported in the literature, we provide a critical review about the scope and limitations of this widely used chemical hypoxia model to be informative to all researchers interested in the field.

Cancer-associated isocitrate dehydrogenase mutations induce mitochondrial DNA instability.

A major advance in understanding the progression and prognostic outcome of certain cancers, such as low-grade gliomas, acute myeloid leukaemia, and chondrosarcomas, has been the identification of early-occurring mutations in the NADP+-dependent isocitrate dehydrogenase genes IDH1 and IDH2 These mutations result in the production of the onco-metabolite D-2-hydroxyglutarate (2HG), thought to contribute to disease progression. To better understand the mechanisms of 2HG pathophysiology, we introduced the analogous glioma-associated mutations into the NADP+ isocitrate dehydrogenase genes (IDP1, IDP2, IDP3) in Saccharomyces cerevisiae Intriguingly, expression of the mitochondrial IDP1R148H mutant allele results in high levels of 2HG production as well as extensive mtDNA loss and respiration defects. We find no evidence for a reactive oxygen-mediated mechanism mediating this mtDNA loss. Instead, we show that 2HG production perturbs the iron sensing mechanisms as indicated by upregulation of the Aft1-controlled iron regulon and a concomitant increase in iron levels. Accordingly, iron chelation, or overexpression of a truncated AFT1 allele that dampens transcription of the iron regulon, suppresses the loss of respirative capacity. Additional suppressing factors include overexpression of the mitochondrial aldehyde dehydrogenase gene ALD5 or disruption of the retrograde response transcription factor RTG1 Furthermore, elevated α-ketoglutarate levels also suppress 2HG-mediated respiration loss; consistent with a mechanism by which 2HG contributes to mtDNA loss by acting as a toxic α-ketoglutarate analog. Our findings provide insight into the mechanisms that may contribute to 2HG oncogenicity in glioma and acute myeloid leukaemia progression, with the promise for innovative diagnostic and prognostic strategies and novel therapeutic modalities.

Calcineurin Targets Involved in Stress Survival and Fungal Virulence.

Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Calcineurin is activated by increased Ca2+ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO2 enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in Saccharomyces cerevisiae. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet to be characterized as calcineurin targets in other organisms. These findings further highlight C. neoformans as an outstanding model to define calcineurin-responsive virulence networks as targets for antifungal therapy.

Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae.

The conserved target of rapamycin complex 1 (TORC1) integrates nutrient signals to orchestrate cell growth and proliferation. Leucine availability is conveyed to control TORC1 activity via the leu-tRNA synthetase/EGOC-GTPase module in yeast and mammals, but the mechanisms sensing leucine remain only partially understood. We show here that both leucine and its α-ketoacid metabolite, α-ketoisocaproate, effectively activate the yeast TORC1 kinase via both EGOC GTPase-dependent and -independent mechanisms. Leucine and α-ketoisocaproate are interconverted by ubiquitous branched-chain aminotransferases (BCAT), which in yeast are represented by the mitochondrial and cytosolic enzymes Bat1 and Bat2, respectively. BCAT yeast mutants exhibit severely compromised TORC1 activity, which is partially restored by expression of Bat1 active site mutants, implicating both catalytic and structural roles of BCATs in TORC1 control. We find that Bat1 interacts with branched-chain amino acid metabolic enzymes and, in a leucine-dependent fashion, with the tricarboxylic acid (TCA)-cycle enzyme aconitase. BCAT mutation perturbed TCA-cycle intermediate levels, consistent with a TCA-cycle block, and resulted in low ATP levels, activation of AMPK, and TORC1 inhibition. We propose the biosynthetic capacity of BCAT and its role in forming multicomplex metabolons connecting branched-chain amino acids and TCA-cycle metabolism governs TCA-cycle flux to activate TORC1 signaling. Because mammalian mitochondrial BCAT is known to form a supramolecular branched-chain α-keto acid dehydrogenase enzyme complex that links leucine metabolism to the TCA-cycle, these findings establish a precedent for understanding TORC1 signaling in mammals.

L-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome.

Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS.

Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor.

The zygomycete Mucor circinelloides is an opportunistic fungal pathogen that commonly infects patients with malignancies, diabetes mellitus, and solid organ transplants. Despite the widespread use of antifungal therapy in the management of zygomycosis, the incidence of infections continues to rise among immunocompromised individuals. In this study, we established that the target and mechanism of antifungal action of the immunosuppressant rapamycin in M. circinelloides are mediated via conserved complexes with FKBP12 and a Tor homolog. We found that spontaneous mutations that disrupted conserved residues in FKBP12 conferred rapamycin and FK506 resistance. Disruption of the FKBP12-encoding gene, fkbA, also conferred rapamycin and FK506 resistance. Expression of M. circinelloides FKBP12 (McFKBP12) complemented a Saccharomyces cerevisiae mutant strain lacking FKBP12 to restore rapamycin sensitivity. Expression of the McTor FKBP12-rapamycin binding (FRB) domain conferred rapamycin resistance in S. cerevisiae, and McFKBP12 interacted in a rapamycin-dependent fashion with the McTor FRB domain in a yeast two-hybrid assay, validating McFKBP12 and McTor as conserved targets of rapamycin. We showed that in vitro, rapamycin exhibited potent growth inhibitory activity against M. circinelloides. In a Galleria mellonella model of systemic mucormycosis, rapamycin improved survival by 50%, suggesting that rapamycin and nonimmunosuppressive analogs have the potential to be developed as novel antifungal therapies for treatment of patients with mucormycosis.

Calcineurin colocalizes with P-bodies and stress granules during thermal stress in Cryptococcus neoformans.

Calcineurin is a calcium-calmodulin-activated serine/threonine-specific phosphatase that operates during cellular responses to stress and plays a prominent role in transcriptional control, whereas regulatory events beyond transcription are less well characterized. This study reveals a novel transcription-independent role of calcineurin during the temperature stress response in the human fungal pathogen Cryptococcus neoformans. The diffusely cytoplasmic calcineurin catalytic subunit Cna1 relocates to endoplasmic reticulum (ER)-associated puncta and the mother-bud neck when cells are subjected to 37°C. More than 50% of Cna1 puncta contain the P-body constituent decapping enzyme Dcp1 and the stress granule constituent poly(A)-binding protein Pub1. These results support a model in which calcineurin orchestrates thermal stress responses by associating with sites of mRNA processing.

The protein kinase Tor1 regulates adhesin gene expression in Candida albicans.

Eukaryotic cell growth is coordinated in response to nutrient availability, growth factors, and environmental stimuli, enabling cell-cell interactions that promote survival. The rapamycin-sensitive Tor1 protein kinase, which is conserved from yeasts to humans, participates in a signaling pathway central to cellular nutrient responses. To gain insight into Tor-mediated processes in human fungal pathogens, we have characterized Tor signaling in Candida albicans. Global transcriptional profiling revealed evolutionarily conserved roles for Tor1 in regulating the expression of genes involved in nitrogen starvation responses and ribosome biogenesis. Interestingly, we found that in C. albicans Tor1 plays a novel role in regulating the expression of several cell wall and hyphal specific genes, including adhesins and their transcriptional repressors Nrg1 and Tup1. In accord with this transcriptional profile, rapamycin induced extensive cellular aggregation in an adhesin-dependent fashion. Moreover, adhesin gene induction and cellular aggregation of rapamycin-treated cells were strongly dependent on the transactivators Bcr1 and Efg1. These findings support models in which Tor1 negatively controls cellular adhesion by governing the activities of Bcr1 and Efg1. Taken together, these results provide evidence that Tor1-mediated cellular adhesion might be broadly conserved among eukaryotic organisms.

Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has developed specialized mechanisms that enable growth on suboptimal nitrogen sources. Exposure of yeast cells to poor nitrogen sources or treatment with the Tor kinase inhibitor rapamycin elicits activation of Gln3 and transcription of nitrogen catabolite-repressed (NCR) genes whose products function in scavenging and metabolizing nitrogen. Here, we show that mutations in class C and D Vps components, which mediate Golgi-to-endosome vesicle transport, impair nuclear translocation of Gln3, NCR gene activation, and growth in poor nitrogen sources. In nutrient-replete conditions, a significant fraction of Gln3 is peripherally associated with light membranes and partially colocalizes with Vps10-containing foci. These results reveal a role for Golgi-to-endosome vesicular trafficking in TORC1-controlled nuclear translocation of Gln3 and support a model in which Tor-mediated signaling in response to nutrient cues occurs in these compartments. These findings have important implications for nutrient sensing and growth control via mTor pathways in metazoans.

Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom.

BACKGROUND: The nutrient-sensing Tor pathway governs cell growth and is conserved in nearly all eukaryotic organisms from unicellular yeasts to multicellular organisms, including humans. Tor is the target of the immunosuppressive drug rapamycin, which in complex with the prolyl isomerase FKBP12 inhibits Tor functions. Rapamycin is a gold standard drug for organ transplant recipients that was approved by the FDA in 1999 and is finding additional clinical indications as a chemotherapeutic and antiproliferative agent. Capitalizing on the plethora of recently sequenced genomes we have conducted comparative genomic studies to annotate the Tor pathway throughout the fungal kingdom and related unicellular opisthokonts, including Monosiga brevicollis, Salpingoeca rosetta, and Capsaspora owczarzaki. RESULTS: Interestingly, the Tor signaling cascade is absent in three microsporidian species with available genome sequences, the only known instance of a eukaryotic group lacking this conserved pathway. The microsporidia are obligate intracellular pathogens with highly reduced genomes, and we hypothesize that they lost the Tor pathway as they adapted and streamlined their genomes for intracellular growth in a nutrient-rich environment. Two TOR paralogs are present in several fungal species as a result of either a whole genome duplication or independent gene/segmental duplication events. One such event was identified in the amphibian pathogen Batrachochytrium dendrobatidis, a chytrid responsible for worldwide global amphibian declines and extinctions. CONCLUSIONS: The repeated independent duplications of the TOR gene in the fungal kingdom might reflect selective pressure acting upon this kinase that populates two proteinaceous complexes with different cellular roles. These comparative genomic analyses illustrate the evolutionary trajectory of a central nutrient-sensing cascade that enables diverse eukaryotic organisms to respond to their natural environments.

Nutritional control via Tor signaling in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae senses and responds to nutrients by adapting its growth rate and undergoing morphogenic transitions to ensure survival. The Tor pathway is a major integrator of nutrient-derived signals that in coordination with other signaling pathways orchestrates cell growth. Recent advances have identified novel Tor kinase substrates and established the protein trafficking membranous network and the nucleus as platforms for Tor signaling. These and other recent findings delineate distinct signaling branches emanating from membrane-associated Tor complexes to control cell growth.

Calcium-Calmodulin-Calcineurin Signaling: A Globally Conserved Virulence Cascade in Eukaryotic Microbial Pathogens.

Calcium is an abundant intracellular ion, and calcium homeostasis plays crucial roles in several cellular processes. The calcineurin signaling cascade is one of the major pathways governed by intracellular calcium. Calcineurin, a conserved protein from yeast to humans, is a calcium-calmodulin-dependent serine-threonine-specific phosphatase that orchestrates cellular stress responses. In eukaryotic microbial pathogens, calcineurin controls essential virulence pathways, such as the ability to grow at host temperature, morphogenesis to enable invasive hyphal growth, drug tolerance and resistance, cell wall integrity, and sexual development. Therefore, the calcineurin cascade is an attractive target in drug development against eukaryotic pathogens. In the present review, we summarize and discuss the current knowledge on the roles of calcineurin in eukaryotic microbial pathogens, focusing on fungi and parasitic protists.

Pbp1-Interacting Protein Mkt1 Regulates Virulence and Sexual Reproduction in Cryptococcus neoformans.

The Mkt1-Pbp1 complex promotes mating-type switching by regulating the translation of HO mRNA in Saccharomyces cerevisiae. Here, we performed in vivo immunoprecipitation assays and mass spectrometry analyses in the human fungal pathogen Cryptococcus neoformans to show that Pbp1, a poly(A)-binding protein-binding protein, interacts with Mkt1 containing a PIN like-domain. Association of Pbp1 with Mkt1 was confirmed by co-immunoprecipitation assays. Results of spot dilution growth assays showed that unlike pbp1 deletion mutant strains, mkt1 deletion mutant strains were not resistant to heat stress compared with wild-type. However, similar to the pbp1 deletion mutant strains, the mkt1 deletion mutants exhibited both, defective dikaryotic hyphal production and reduced pheromone gene (MFα1) expression during mating. In addition, deletion of mkt1 caused attenuated virulence in a murine intranasal inhalation model. Taken together, our findings reveal that Mkt1 plays a crucial role in sexual reproduction and virulence in C. neoformans.

Diallyl Trisulfide Protects Rat Brain Tissue against the Damage Induced by Ischemia-Reperfusion through the Nrf2 Pathway.

Stroke is a public health problem due to its high mortality and disability rates; despite these, the pharmacological treatments are limited. Oxidative stress plays an important role in cerebral damage in stroke and the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) confers protection against oxidative stress. Different compounds, such as diallyl trisulfide (DATS), have the ability to activate Nrf2. DATS protects against the damage induced in oxygen-glucose deprivation in neuronal cells; however, in in vivo models of cerebral ischemia, DATS has not been evaluated. Male Wistar rats were subjected to 1 h of ischemia and seven days of reperfusion and the protective effect of DATS was evaluated. DATS administration (IR + DATS) decreased the infarct area and brain damage in the striatum and cortex; improved neurological function; decreased malondialdehyde and metalloproteinase-9 levels; increased Nrf2 activation in the cortex and the expression of superoxide dismutase 1 (SOD1) in the nucleus, SOD2 and glutathione S-transferase (GST) in the striatum and cortex; and increased the activity of catalase (CAT) in the striatum and glutathione peroxidase (GPx) in the cortex. Our results demonstrate the protective effect of DATS in an in vivo model of cerebral ischemia that involves Nrf2 activation.

The conserved salt bridge linking two C-terminal beta/alpha units in homodimeric triosephosphate isomerase determines the folding rate of the monomer.

Triosephosphate isomerase (TIM), whose structure is archetypal of dimeric (beta/alpha)(8) barrels, has a conserved salt bridge (Arg189-Asp225 in yeast TIM) that connects the two C-terminal beta/alpha segments to rest of the monomer. We constructed the mutant D225Q, and studied its catalysis and stability in comparison with those of the wild-type enzyme. Replacement of Asp225 by Gln caused minor drops in k(cat) and K(M), but the catalytic efficiency (k(cat)/K(M)) was practically unaffected. Temperature-induced unfolding-refolding of both TIM samples displayed hysteresis cycles, indicative of processes far from equilibrium. Kinetic studies showed that the rate constant for unfolding was about three-fold larger in the mutant than in wild-type TIM. However, more drastic changes were found in the kinetics of refolding: upon mutation, the rate-limiting step changed from a second-order (at submicromolar concentrations) to a first-order reaction. These results thus indicate that renaturation of yTIM occurs through a uni-bimolecular mechanism in which refolding of the monomer most likely begins at the C-terminal half of its polypeptide chain. From the temperature dependence of the refolding rate, we determined the change in heat capacity for the formation of the transition state from unfolded monomers. The value for the D225Q mutant, which is about 40% of the corresponding value for yTIM, would implicate the folding of only three quarters of a monomer chain in the transition state.