Browsing by Subject "Glutathione"

Now showing 1 - 5 of 5
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    Coxiella burnetii Virulent Phase I and Avirulent Phase II Variants Differentially Manipulate Autophagy Pathway in Neutrophils
    (American Society for Microbiology, 2022) Kumaresan, Venkatesh; Wang, Juexin; Zhang, Wendy; Zhang, Yan; Xu, Dong; Zhang, Guoquan; Medical and Molecular Genetics, School of Medicine
    Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes Q fever in humans. The virulent C. burnetii Nine Mile phase I (NMI) strain causes disease in animal models, while the avirulent NM phase II (NMII) strain does not. In this study, we found that NMI infection induces severe splenomegaly and bacterial burden in the spleen in BALB/c mice, while NMII infection does not. A significantly higher number of CD11b+ Ly6G+ neutrophils accumulated in the liver, lung, and spleen of NMI-infected mice than in NMII-infected mice. Thus, neutrophil accumulation correlates with NMI and NMII infection-induced inflammatory responses. In vitro studies also demonstrated that although NMII exhibited a higher infection rate than NMI in mouse bone marrow neutrophils (BMNs), NMI-infected BMNs survived longer than NMII-infected BMNs. These results suggest that the differential interactions of NMI and NMII with neutrophils may be related to their ability to cause disease in animals. To understand the molecular mechanism underlying the differential interactions of NMI and NMII with neutrophils, global transcriptomic gene expressions were compared between NMI- and NMII-infected BMNs by RNA sequencing (RNA-seq) analysis. Interestingly, several genes involved in autophagy-related pathways, particularly membrane trafficking and lipid metabolism, are upregulated in NMII-infected BMNs but downregulated in NMI-infected BMNs. Immunofluorescence and immunoblot analyses indicate that compared to NMI-infected BMNs, vacuoles in NMII-infected-BMNs exhibit increased autophagic flux along with phosphatidylserine translocation in the cell membrane. Similar to neutrophils, NMII activated LC3-mediated autophagy in human macrophages. These findings suggest that the differential manipulation of autophagy of NMI and NMII may relate to their pathogenesis.
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    Evidence Favoring a Positive Feedback Loop for Physiologic Auto Upregulation of hnRNP-E1 during Prolonged Folate Deficiency in Human Placental Cells
    (Oxford University Press, 2017-04) Tang, Ying-Sheng; Khan, Rehana A.; Xiao, Suhong; Hansen, Deborah K.; Stabler, Sally P.; Kusumanchi, Praveen; Jayaram, Hiremagalur N.; Antony, Aśok C.; Medicine, School of Medicine
    Background: Previously, we determined that heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) functions as an intracellular physiologic sensor of folate deficiency. In this model, l-homocysteine, which accumulates intracellularly in proportion to the extent of folate deficiency, covalently binds to and thereby activates homocysteinylated hnRNP-E1 to interact with folate receptor-α mRNA; this high-affinity interaction triggers the translational upregulation of cell surface folate receptors, which enables cells to optimize folate uptake from the external milieu. However, integral to this model is the need for ongoing generation of hnRNP-E1 to replenish homocysteinylated hnRNP-E1 that is degraded.Objective: We searched for an interrelated physiologic mechanism that could also maintain the steady-state concentration of hnRNP-E1 during prolonged folate deficiency.Methods: A novel RNA-protein interaction was functionally characterized by using molecular and biochemical approaches in vitro and in vivo.Results: l-homocysteine triggered a dose-dependent high-affinity interaction between hnRNP-E1 and a 25-nucleotide cis element within the 5'-untranslated region of hnRNP-E1 mRNA; this led to a proportionate increase in these RNA-protein complexes, and translation of hnRNP-E1 both in vitro and within placental cells. Targeted perturbation of this RNA-protein interaction either by specific 25-nucleotide antisense oligonucleotides or mutation within this cis element or by small interfering RNA to hnRNP-E1 mRNA significantly reduced cellular biosynthesis of hnRNP-E1. Conversely, transfection of hnRNP-E1 mutant proteins that mimicked homocysteinylated hnRNP-E1 stimulated both cellular hnRNP-E1 and folate receptor biosynthesis. In addition, ferrous sulfate heptahydrate [iron(II)], which also binds hnRNP-E1, significantly perturbed this l-homocysteine-triggered RNA-protein interaction in a dose-dependent manner. Finally, folate deficiency induced dual upregulation of hnRNP-E1 and folate receptors in cultured human cells and tumor xenografts, and more selectively in various fetal tissues of folate-deficient dams.Conclusions: This novel positive feedback loop amplifies hnRNP-E1 during prolonged folate deficiency and thereby maximizes upregulation of folate receptors in order to restore folate homeostasis toward normalcy in placental cells. It will also functionally impact several other mRNAs of the nutrition-sensitive, folate-responsive posttranscriptional RNA operon that is orchestrated by homocysteinylated hnRNP-E1.
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    Investigation of the potential bacterial proteasome homologue Anbu
    (2014-09-08) Suknaic, Stephen R.; Kusmierczyk, Andrew; Randall, Stephen Karl, 1953-; Anderson, Gregory G.
    Anbu is a bacterial protein with significant homology to the sub-units of the 20S proteasome and is predicted to be a novel bacterial proteasome. The goal of this project was to determine if the recombinant Anbu protein from Pseudomonas aeruginosa is a proteasome. Anbu from P. aeruginosa was successfully cloned, expressed and purified. In order to determine the catalytic activity of Anbu, the purified protein was tested with a variety of substrates and conditions. The targets analyzed included fluorescently-labeled substrates, denatured proteins, diubiquitin, and a peptide library in the hopes of obtaining a useful model substrate. Experiments were also conducted to determine what role Anbu has in the cell. Western analysis was performed on the cell lysate of wild type P. aeruginosa and insertional mutants to detect Anbu expression. The level of biofilm formation was compared between the wild type and mutants. Cultures were grown under stress conditions including the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione. Growth rates were monitored in an attempt to detect a phenotypic difference between the wild type and the mutants lacking Anbu, HslV, and the other proteins of interest. While a substrate for Anbu has yet to be found, this protein was found to assemble into a larger structure and P. aeruginosa lacking Anbu was sensitive to the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione.
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    Personalized Genome-Scale Metabolic Models Identify Targets of Redox Metabolism in Radiation-Resistant Tumors
    (Cell Press, 2021) Lewis, Joshua E.; Forshaw, Tom E.; Boothman, David A.; Furdui, Cristina M.; Kemp, Melissa L.; Biochemistry and Molecular Biology, School of Medicine
    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.
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    Using substantial reductant concentration with chelation therapy to enhance small aggregate dispersal, iron mobilization, and its clearance in neurodegenerative diseases
    (Frontiers Media, 2022-09-15) Muhoberac, Barry B.; Chemistry and Chemical Biology, School of Science
    Connections between altered iron homeostasis and certain neurodegenerative diseases are highlighted by numerous studies suggesting iron neurotoxicity. Iron causes aggregation in neurodegenerative disease-linked proteins as well as others and additionally facilitates oxidative damage. Iron and oxidative damage can cause cell death including by ferroptosis. As treatment for neurodegeneration, chelation therapy alone is sometimes used with modest, varying efficacy and has not in general proven to reverse or halt the damage long term. Questions often focus on optimal chelator partitioning and fine-tuning binding strength; however iron oxidation state chemistry implies a different approach. More specifically, my perspective is that applying a redox-based component to iron mobilization and handling is crucial because ferrous iron is in general a more soluble, weaker biological binder than ferric. Once cellular iron becomes oxidized to ferric, it binds tenaciously, exchanges ligands more slowly, and enhances protein aggregation, which importantly can be reversed by iron reduction. This situation escalates with age as brain reducing ability decreases, iron concentration increases, autophagic clearance decreases, and cell stress diminishes iron handling capacity. Taken together, treatment employing chelation therapy together with a strong biological reductant may effectively remove inappropriately bound cellular iron or at least inhibit accumulation. This approach would likely require high concentration ascorbate or glutathione by IV along with chelation to enhance iron mobilization and elimination, thus reducing cumulative cellular damage and perhaps restoring partial function. Potential treatment-induced oxidative damage may be attenuated by high reductant concentration, appropriate choice of chelator, and/or treatment sequence. Comprehensive study is urged.