Reduction of ribonuclease by glutathione at elevated temperatures: the molecular mechanism.
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ABSTRACT:
SUBMITTER: Davidson BE
PROVIDER: S-EPMC1270609 | biostudies-other | 1967 Aug
REPOSITORIES: biostudies-other
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Project description:Glutathionyl-hydroquinone reductases (GS- HQRs) are a newly identified group of glutathione transferases, and they are widely distributed in bacteria, halobacteria, fungi, and plants. GS-HQRs catalyze glutathione (GSH)-dependent reduction of glutathionyl-hydroquinones (GS-hydroquinones) to hydroquinones. GS-hydroquinones can be spontaneously formed from benzoquinones reacting with reduced GSH via Michael addition, and GS-HQRs convert the conjugates to hydroquinones. In this report we have determined the structures of two bacterial GS-HQRs, PcpF of Sphingobium chlorophenolicum and YqjG of Escherichia coli. The two structures and the previously reported structure of a fungal GS-HQR shared many features and displayed complete conservation for all the critical residues. Furthermore, we obtained the binary complex structures with GS-menadione, which in its reduced form, GS-menadiol, is a substrate. The structure revealed a large H-site that could accommodate various substituted hydroquinones and a hydrogen network of three Tyr residues that could provide the proton for reductive deglutathionylation. Mutation of the Tyr residues and the position of two GSH molecules confirmed the proposed mechanism of GS-HQRs. The conservation of GS-HQRs across bacteria, halobacteria, fungi, and plants potentiates the physiological role of these enzymes in quinone metabolism.
| S-EPMC3476253 | biostudies-literature
Project description:In light of global climate change, there is a pressing need to understand and predict the capacity of populations to respond to rising temperatures. Metabolic rate is a key trait that is likely to influence the ability to cope with climate change. Yet, empirical and theoretical work on metabolic rate responses to temperature changes has so far produced mixed results and conflicting predictions.Our study addresses this issue using a novel approach of comparing fish populations in geothermally warmed lakes and adjacent ambient-temperature lakes in Iceland. This unique 'natural experiment' provides repeated and independent examples of populations experiencing contrasting thermal environments for many generations over a small geographic scale, thereby avoiding the confounding factors associated with latitudinal or elevational comparisons. Using Icelandic sticklebacks from three warm and three cold habitats, we measured individual metabolic rates across a range of acclimation temperatures to obtain reaction norms for each population.We found a general pattern for a lower standard metabolic rate (SMR) in sticklebacks from warm habitats when measured at a common temperature, as predicted by Krogh's rule. Metabolic rate differences between warm- and cold-habitat sticklebacks were more pronounced at more extreme acclimation temperatures, suggesting the release of cryptic genetic variation upon exposure to novel conditions, which can reveal hidden evolutionary potential. We also found a stronger divergence in metabolic rate between thermal habitats in allopatry than sympatry, indicating that gene flow may constrain physiological adaptation when dispersal between warm and cold habitats is possible.In sum, our study suggests that fish may diverge toward a lower SMR in a warming world, but this might depend on connectivity and gene flow between different thermal habitats. A free Plain Language Summary can be found within the Supporting Information of this article.
| S-EPMC7318562 | biostudies-literature
Project description:Our recent findings show that astrocyte elevated gene-1 (AEG-1) is overexpressed in >90% of human hepatocellular carcinoma (HCC) samples, and AEG-1 plays a central role in regulating development and progression of HCC. In the present study, we elucidate a molecular mechanism of AEG-1-induced chemoresistance, an important characteristic of aggressive cancers. AEG-1 increases the expression of multidrug resistance gene 1 (MDR1) protein, resulting in increased efflux and decreased accumulation of doxorubicin, promoting doxorubicin resistance. Suppression of MDR1 by small interfering RNA or chemical reagents, or inhibition of AEG-1 or a combination of both genes, significantly increases in vitro sensitivity to doxorubicin. In nude mice xenograft studies, a lentivirus expressing AEG-1 short hairpin RNA, in combination with doxorubicin, profoundly inhibited growth of aggressive human HCC cells compared with either agent alone. We document that although AEG-1 does not affect MDR1 gene transcription, it facilitates association of MDR1 mRNA to polysomes, resulting in increased translation, and AEG-1 also inhibits ubiquitination and subsequent proteasome-mediated degradation of MDR1 protein. This study is the first documentation of a unique aspect of AEG-1 function (i.e., translational and posttranslational regulation of proteins). Inhibition of AEG-1 might provide a means of more effectively using chemotherapy to treat HCC, which displays inherent chemoresistance with aggressive pathology.
| S-EPMC2855753 | biostudies-literature
Project description:Type III CRISPR systems are innate immune systems found in bacteria and archaea, which produce cyclic oligoadenylate (cOA) second messengers in response to viral infections. In these systems, Csm6 proteins serve as ancillary nucleases that degrade single-stranded RNA (ssRNA) upon activation by cOA. In addition, Csm6 proteins also possess cOA-degrading activity as an intrinsic off-switch to avoid degradation of host RNA and DNA that would eventually lead to cell dormancy or cell death. Here, we present the crystal structures of Thermus thermophilus (Tt) Csm6 alone, and in complex with cyclic tetra-adenylate (cA4) in both pre- and post-cleavage states. These structures establish the molecular basis of the long-range allosteric activation of TtCsm6 ribonuclease by cA4. cA4 binding induces significant conformational changes, including closure of the CARF domain, dimerization of the HTH domain, and reorganization of the R-X4-6-H motif within the HEPN domain. The cleavage of cA4 by the CARF domain restores each domain to a conformation similar to its apo state. Furthermore, we have identified hyperactive TtCsm6 variants that exhibit sustained cA4-activated RNase activity, showing great promise for their applications in genome editing and diagnostics.
| S-EPMC10897365 | biostudies-literature
Project description:BackgroundThe causes of stillbirth are poorly understood, including whether elevated outdoor temperatures increase risk. We assessed the relationship between elevated ambient temperatures and risk of stillbirth by gestational age and cause of death during warm months in a temperate region.MethodsWe performed a case-crossover study of 5047 stillbirths in continental Quebec, Canada, between the months of April through September from 1981 to 2011. Using data on maximum daily temperatures adjusted for relative humidity, we estimated associations with stillbirth, comparing temperatures before fetal death with temperatures on adjacent days. The main outcomes were stillbirth according to age of gestation (term, preterm), and cause of death (undetermined, maternal, placenta/cord/membranes, birth asphyxia, congenital anomaly, other).ResultsElevated outdoor temperatures the week before the death were more strongly associated with risk of term than preterm stillbirth. Odds of term stillbirth for temperature 28?°C the day before death were 1.16 times greater relative to 20?°C (95% confidence interval, CI 1.02-1.33). Elevated outdoor temperature was associated with stillbirth due to undetermined and maternal causes, but not other causes. Compared with 20?°C, the odds of stillbirth at 28?°C were 1.19 times greater for undetermined causes (95% CI 1.02-1.40) and 1.46 times greater for maternal complications (95% CI 1.03-2.07).ConclusionsElevated outdoor temperatures may be a risk factor for term stillbirth, including stillbirth due to undetermined causes or maternal complications.
| S-EPMC5837696 | biostudies-literature
Project description:The conventional view is that high temperatures cause microorganisms to replicate slowly or die. In this view, microorganisms autonomously combat heat-induced damage. However, microorganisms co-exist with each other, which raises the underexplored and timely question of whether microorganisms can cooperatively combat heat-induced damages at high temperatures. Here, we use the budding yeast Saccharomyces cerevisiae to show that cells can help each other and their future generations to survive and replicate at high temperatures. As a consequence, even at the same temperature, a yeast population can exponentially grow, never grow or grow after unpredictable durations (hours to days) of stasis, depending on its population density. Through the same mechanism, yeasts collectively delay and can eventually stop their approach to extinction, with higher population densities stopping faster. These features arise from yeasts secreting and extracellularly accumulating glutathione—a ubiquitous heat-damage-preventing antioxidant. We show that the secretion of glutathione, which eliminates harmful extracellular chemicals, is both necessary and sufficient for yeasts to collectively survive at high temperatures. A mathematical model, which is generally applicable to any cells that cooperatively replicate by secreting molecules, recapitulates all of these features. Our study demonstrates how organisms can cooperatively define and extend the boundaries of life-permitting temperatures.
2020-03-24 | GSE137151 | GEO
Project description:GSH, but not GSSG, inhibits the reactivation by phosphate ion of ribonuclease activity inactivated by urea or guanidine. The effects of GSH are rather slow and pretreatment of ribonuclease with urea is a requisite for the inhibitory action of GSH on enzyme reactivation. GSH is more effective in urea than in guanidine and its action is greatly enhanced by EDTA. An optimum pH of about 9.0 was found for the inhibitory effect of GSH. Titration of the thiol groups formed after inactivation of ribonuclease by GSH strongly suggests that the reduction of only one disulphide linkage is involved. The reduction of this bond is sufficient to completely abolish the enzymic activity.
| S-EPMC1198562 | biostudies-other
Project description:We use quantum mechanics/molecular mechanics simulations to study the cleavage of the ribonucleic acid (RNA) backbone catalyzed by ribonuclease H. This protein is a prototypical member of a large family of enzymes that use two-metal catalysis to process nucleic acids. By combining Hamiltonian replica exchange with a finite-temperature string method, we calculate the free energy surface underlying the RNA-cleavage reaction and characterize its mechanism. We find that the reaction proceeds in two steps. In a first step, catalyzed primarily by magnesium ion A and its ligands, a water molecule attacks the scissile phosphate. Consistent with thiol-substitution experiments, a water proton is transferred to the downstream phosphate group. The transient phosphorane formed as a result of this nucleophilic attack decays by breaking the bond between the phosphate and the ribose oxygen. In the resulting intermediate, the dissociated but unprotonated leaving group forms an alkoxide coordinated to magnesium ion B. In a second step, the reaction is completed by protonation of the leaving group, with a neutral Asp132 as a likely proton donor. The overall reaction barrier of ∼15 kcal mol(-1), encountered in the first step, together with the cost of protonating Asp132, is consistent with the slow measured rate of ∼1-100/min. The two-step mechanism is also consistent with the bell-shaped pH dependence of the reaction rate. The nonmonotonic relative motion of the magnesium ions along the reaction pathway agrees with X-ray crystal structures. Proton-transfer reactions and changes in the metal ion coordination emerge as central factors in the RNA-cleavage reaction.
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Project description:Nuclear-grade graphite is a critically important high-temperature structural material for current and potentially next generation of fission reactors worldwide. It is imperative to understand its damage-tolerant behaviour and to discern the mechanisms of damage evolution under in-service conditions. Here we perform in situ mechanical testing with synchrotron X-ray computed micro-tomography at temperatures between ambient and 1,000?°C on a nuclear-grade Gilsocarbon graphite. We find that both the strength and fracture toughness of this graphite are improved at elevated temperature. Whereas this behaviour is consistent with observations of the closure of microcracks formed parallel to the covalent-sp2-bonded graphene layers at higher temperatures, which accommodate the more than tenfold larger thermal expansion perpendicular to these layers, we attribute the elevation in strength and toughness primarily to changes in the residual stress state at 800-1,000?°C, specifically to the reduction in significant levels of residual tensile stresses in the graphite that are 'frozen-in' following processing.
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Project description:In
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