Homozygous knockout of eEF2K alleviates cognitive deficits in APP/PS1 Alzheimer's disease model mice independent of brain amyloid ß pathology.

Maintenance of memory and synaptic plasticity depends on de novo protein synthesis, and accumulating evidence implicates a role of dysregulated mRNA translation in cognitive impairments associated with Alzheimer's disease (AD). Accumulating evidence demonstrates hyper-phosphorylation of translation factor eukaryotic elongation factor 2 (eEF2) in the hippocampi of human AD patients as well as transgenic AD model mice. Phosphorylation of eEF2 (at the Thr 56 site) by its only known kinase, eEF2K, leads to inhibition of general protein synthesis. A recent study suggests that amyloid ß (Aß)-induced neurotoxicity could be associated with an interaction between eEF2 phosphorylation and the transcription factor nuclear erythroid 2-related factor (NRF2)-mediated antioxidant response. In this brief communication, we report that global homozygous knockout of the eEF2K gene alleviates deficits of long-term recognition and spatial learning in a mouse model of AD (APP/PS1). Moreover, eEF2K knockout does not alter brain Aß pathology in APP/PS1 mice. The hippocampal NRF2 antioxidant response in the APP/PS1 mice, measured by expression levels of nicotinamide adenine dinucleotide plus hydrogen (NADPH) quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), is ameliorated by suppression of eEF2K signaling. Together, the findings may contribute to our understanding of the molecular mechanisms underlying AD pathogenesis, indicating that suppression of eEF2K activity could be a beneficial therapeutic option for this devastating neurodegenerative disease.

Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition.

The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pKa of the C12 thiol is depressed (pKa ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, Kd = 36.0 ± 0.7 µM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.

[18F]Fluoro-DCP, a first generation PET radiotracer for monitoring protein sulfenylation in vivo.

Redox metabolism plays essential functions in the pathology of cancer and many other diseases. While several radiotracers for imaging redox metabolism have been developed, there are no reports of radiotracers for in vivo imaging of protein oxidation. Here we take the first step towards this goal and describe the synthesis and kinetic properties of a new positron emission tomography (PET) [18F]Fluoro-DCP radiotracer for in vivo imaging of protein sulfenylation. Time course biodistribution and PET/CT studies using xenograft animal models of Head and Neck Squamous Cell Cancer (HNSCC) demonstrate its capability to distinguish between tumors with radiation sensitive and resistant phenotypes consistent with previous reports of decreased protein sulfenylation in clinical specimens of radiation resistant HNSCC. We envision further development of this technology to aid research efforts towards improving diagnosis of patients with radiation resistant tumors.

Specificity of Human Sulfiredoxin for Reductant and Peroxiredoxin Oligomeric State.

Human peroxiredoxins (Prx) are a family of antioxidant enzymes involved in a myriad of cellular functions and diseases. During the reaction with peroxides (e.g., H2O2), the typical 2-Cys Prxs change oligomeric structure between higher order (do)decamers and disulfide-linked dimers, with the hyperoxidized inactive state (-SO2H) favoring the multimeric structure of the reduced enzyme. Here, we present a study on the structural requirements for the repair of hyperoxidized 2-Cys Prxs by human sulfiredoxin (Srx) and the relative efficacy of physiological reductants hydrogen sulfide (H2S) and glutathione (GSH) in this reaction. The crystal structure of the toroidal Prx1-Srx complex shows an extended active site interface. The loss of this interface within engineered Prx2 and Prx3 dimers yielded variants more resistant to hyperoxidation and repair by Srx. Finally, we reveal for the first time Prx isoform-dependent use of and potential cooperation between GSH and H2S in supporting Srx activity.

Personalized Genome-Scale Metabolic Models Identify Targets of Redox Metabolism in Radiation-Resistant Tumors.

Redox cofactor production is integral toward antioxidant generation, clearance of reactive oxygen species, and overall tumor response to ionizing radiation treatment. To identify systems-level alterations in redox metabolism that confer resistance to radiation therapy, we developed a bioinformatics pipeline for integrating multi-omics data into personalized genome-scale flux balance analysis models of 716 radiation-sensitive and 199 radiation-resistant tumors. These models collectively predicted that radiation-resistant tumors reroute metabolic flux to increase mitochondrial NADPH stores and reactive oxygen species (ROS) scavenging. Simulated genome-wide knockout screens agreed with experimental siRNA gene knockdowns in matched radiation-sensitive and radiation-resistant cancer cell lines, revealing gene targets involved in mitochondrial NADPH production, central carbon metabolism, and folate metabolism that allow for selective inhibition of glutathione production and H2O2 clearance in radiation-resistant cancers. This systems approach represents a significant advancement in developing quantitative genome-scale models of redox metabolism and identifying personalized metabolic targets for improving radiation sensitivity in individual cancer patients.

Quantitative analysis of calf mortality in Great Britain.

National bodies in Great Britain (GB) have expressed concern over young stock health and welfare and identified calf survival as a priority; however, no national data have been available to quantify mortality rates. The aim of this study was to quantify the temporal incidence rate, distributional features, and factors affecting variation in mortality rates in calves in GB since 2011. The purpose was to provide information to national stakeholder groups to inform resource allocation both for knowledge exchange and future research. Cattle birth and death registrations from the national British Cattle Movement Service were analyzed to determine rates of both slaughter and on-farm mortality. The number of births and deaths registered between 2011 and 2018 within GB were 21.2 and 21.6 million, respectively. Of the 3.3 million on-farm deaths, 1.8 million occurred before 24 mo of age (54%) and 818,845 (25%) happened within the first 3 mo of age. The on-farm mortality rate was 3.87% by 3 mo of age, remained relatively stable over time, and was higher for male calves (4.32%) than female calves (3.45%). Dairy calves experience higher on farm mortality rates than nondairy (beef) calves in the first 3 mo of life, with 6.00 and 2.86% mortality rates, respectively. The 0- to 3-mo death rate at slaughterhouse for male dairy calves has increased from 17.40% in 2011 to 26.16% in 2018, and has remained low (<0.5%) for female dairy calves and beef calves of both sexes. Multivariate adaptive regression spline models were able to explain a large degree of the variation in mortality rates (coefficient of determination = 96%). Mean monthly environmental temperature and month of birth appeared to play an important role in neonatal on-farm mortality rates, with increased temperatures significantly reducing mortality rates. Taking the optimal month of birth and environmental temperature as indicators of the best possible environmental conditions, maintaining these conditions throughout the year would be expected to result in a reduction in annual 0- to 3-mo mortality of 37,571 deaths per year, with an estimated economic saving of around £11.6 million (USD $15.3 million) per annum. National cattle registers have great potential for monitoring trends in calf mortality and can provide valuable insights to the cattle industry. Environmental conditions play a significant role in calf mortality rates and further research is needed to explore how to optimize conditions to reduce calf mortality rates in GB.

Triphenylphosphonium-Derived Protein Sulfenic Acid Trapping Agents: Synthesis, Reactivity, and Effect on Mitochondrial Function.

Redox-mediated protein modifications control numerous processes in both normal and disease metabolism. Protein sulfenic acids, formed from the oxidation of protein cysteine residues, play a critical role in thiol-based redox signaling. The reactivity of protein sulfenic acids requires their identification through chemical trapping, and this paper describes the use of the triphenylphosphonium (TPP) ion to direct known sulfenic acid traps to the mitochondria, a verified source of cellular reactive oxygen species. Coupling of the TPP group with the 2,4-(dioxocyclohexyl)propoxy (DCP) unit and the bicyclo[6.1.0]nonyne (BCN) group produces two new probes, DCP-TPP and BCN-TPP. DCP-TPP and BCN-TPP react with C165A AhpC-SOH, a model protein sulfenic acid, to form the expected adducts with second-order rate constants of k = 1.1 M-1 s-1 and k = 5.99 M-1 s-1, respectively, as determined by electrospray ionization time-of-flight mass spectrometry. The TPP group does not alter the rate of DCP-TPP reaction with protein sulfenic acid compared to dimedone but slows the rate of BCN-TPP reaction compared to a non-TPP-containing BCN-OH control by 4.6-fold. The hydrophobic TPP group may interact with the protein, preventing an optimal reaction orientation for BCN-TPP. Unlike BCN-OH, BCN-TPP does not react with the protein persulfide, C165A AhpC-SSH. Extracellular flux measurements using A549 cells show that DCP-TPP and BCN-TPP influence mitochondrial energetics, with BCN-TPP producing a drastic decrease in basal respiration, perhaps due to its faster reaction kinetics with sulfenylated proteins. Further control experiments with BCN-OH, TPP-COOH, and dimedone provide strong evidence for mitochondrial localization and accumulation of DCP-TPP and BCN-TPP. These results reveal the compatibility of the TPP group with reactive sulfenic acid probes as a mitochondrial director and support the use of the TPP group in the design of sulfenic acid traps.

Peroxiredoxins in Cancer and Response to Radiation Therapies.

Peroxiredoxins have a long-established cellular function as regulators of redox metabolism by catalyzing the reduction of peroxides (e.g., H2O2, lipid peroxides) with high catalytic efficiency. This activity is also critical to the initiation and relay of both phosphorylation and redox signaling in a broad range of pathophysiological contexts. Under normal physiological conditions, peroxiredoxins protect normal cells from oxidative damage that could promote oncogenesis (e.g., environmental stressors). In cancer, higher expression level of peroxiredoxins has been associated with both tumor growth and resistance to radiation therapies. However, this relationship between the expression of peroxiredoxins and the response to radiation is not evident from an analysis of data in The Cancer Genome Atlas (TCGA) or NCI60 panel of cancer cell lines. The focus of this review is to summarize the current experimental knowledge implicating this class of proteins in cancer, and to provide a perspective on the value of targeting peroxiredoxins in the management of cancer. Potential biases in the analysis of the TCGA data with respect to radiation resistance are also highlighted.