Scholarship & Research
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/1
Browse
11 results
Search Results
Item Characterization of synovial fluid metabolomic phenotypes of cartilage morphological changes associated with osteoarthritis(2019-08) Carlson, Alyssa K.; Rawle, Rachel A.; Wallace, Cameron W.; Brooks, Ellen G.; Adams, Erik; Greenwood, Mark C.; Olmer, Merissa; Lotz, Martin K.; Bothner, Brian; June, Ronald K."Objective Osteoarthritis (OA) is a multifactorial disease with etiological heterogeneity. The objective of this study was to classify OA subgroups by generating metabolomic phenotypes from human synovial fluid. Design: Post mortem synovial fluids (n = 75) were analyzed by high performance-liquid chromatography mass spectrometry (LC-MS) to measure changes in the global metabolome. Comparisons of healthy (grade 0), early OA (grades I-II), and late OA (grades III-IV) donor populations were considered to reveal phenotypes throughout disease progression. Results: Global metabolomic profiles in synovial fluid were distinct between healthy, early OA, and late OA donors. Pathways differentially activated among these groups included structural deterioration, glycerophospholipid metabolism, inflammation, central energy metabolism, oxidative stress, and vitamin metabolism. Within disease states (early and late OA), subgroups of donors revealed distinct phenotypes. Synovial fluid metabolomic phenotypes exhibited increased inflammation (early and late OA), oxidative stress (late OA), or structural deterioration (early and late OA) in the synovial fluid. Conclusion: These results revealed distinct metabolic phenotypes in human synovial fluid, provide insight into pathogenesis, represent novel biomarkers, and can move toward developing personalized interventions for subgroups of OA patients.Item TrxR1, Gsr, and oxidative stress determine hepatocellular carcinoma malignancy(2019-06) McLoughlin, Michael R.; Orlicky, David J.; Prigge, Justin R.; Krishna, Pushya; Talago, Emily A.; Cavigli, Ian R.; Eriksson, Sofi; Miller, Colin G.; Kundert, Jean A.; Sayin, Volkan I.; Sabol, Rachel A.; Heinemann, Joshua; Brandenberger, Luke O.; Iverson, Sonya V.; Bothner, Brian; Papagiannakopoulos, Thales; Shearn, Colin T.; Arner, Elias S. J.; Schmidt, Edward E.Thioredoxin reductase-1 (TrxR1)-, glutathione reductase (Gsr)-, and Nrf2 transcription factor-driven antioxidant systems form an integrated network that combats potentially carcinogenic oxidative damage yet also protects cancer cells from oxidative death. Here we show that although unchallenged wild-type (WT), TrxR1-null, or Gsr-null mouse livers exhibited similarly low DNA damage indices, these were 100-fold higher in unchallenged TrxR1/Gsr–double-null livers. Notwithstanding, spontaneous cancer rates remained surprisingly low in TrxR1/Gsr-null livers. All genotypes, including TrxR1/Gsr-null, were susceptible to N-diethylnitrosamine (DEN)-induced liver cancer, indicating that loss of these antioxidant systems did not prevent cancer cell survival. Interestingly, however, following DEN treatment, TrxR1-null livers developed threefold fewer tumors compared with WT livers. Disruption of TrxR1 in a marked subset of DEN-initiated cancer cells had no effect on their subsequent contributions to tumors, suggesting that TrxR1-disruption does not affect cancer progression under normal care, but does decrease the frequency of DEN-induced cancer initiation. Consistent with this idea, TrxR1-null livers showed altered basal and DEN-exposed metabolomic profiles compared with WT livers. To examine how oxidative stress influenced cancer progression, we compared DEN-induced cancer malignancy under chronically low oxidative stress (TrxR1-null, standard care) vs. elevated oxidative stress (TrxR1/Gsr-null livers, standard care or phenobarbital-exposed TrxR1-null livers). In both cases, elevated oxidative stress was correlated with significantly increased malignancy. Finally, although TrxR1-null and TrxR1/Gsr-null livers showed strong Nrf2 activity in noncancerous hepatocytes, there was no correlation between malignancy and Nrf2 expression within tumors across genotypes. We conclude that TrxR1, Gsr, Nrf2, and oxidative stress are major determinants of liver cancer but in a complex, context-dependent manner.Item Something old, something new, something borrowed; how the thermoacidophilic archaeon Sulfolobus solfataricus responds to oxidative stress(2009-09) Maaty, Walid S.; Wiedenheft, Blake A.; Tarlykov, Pavel V.; Schaff, Nathan; Heinemann, Joshua V.; Robison-Cox, James; Dougherty, Amanda; Blum, Paul; Lawrence, C. Martin; Douglas, Trevor; Young, Mark J.; Bothner, BrianTo avoid molecular damage of biomolecules due to oxidation, all cells have evolved constitutive and responsive systems to mitigate and repair chemical modifications. Archaea have adapted to some of the most extreme environments known to support life, including highly oxidizing conditions. However, in comparison to bacteria and eukaryotes, relatively little is known about the biology and biochemistry of archaea in response to changing conditions and repair of oxidative damage. In this study transcriptome, proteome, and chemical reactivity analyses of hydrogen peroxide (H2O2) induced oxidative stress in Sulfolobus solfataricus (P2) were conducted. Microarray analysis of mRNA expression showed that 102 transcripts were regulated by at least 1.5 fold, 30 minutes after exposure to 30 µM H2O2. Parallel proteomic analyses using two-dimensional differential gel electrophoresis (2D-DIGE), monitored more than 800 proteins 30 and 105 minutes after exposure and found that 18 had significant changes in abundance. A recently characterized ferritin-like antioxidant protein, DPSL, was the most highly regulated species of mRNA and protein, in addition to being post-translationally modified. As expected, a number of antioxidant related mRNAs and proteins were differentially regulated. Three of these, DPSL, superoxide dismutase, and peroxiredoxin were shown to interact and likely form a novel supramolecular complex for mitigating oxidative damage. A scheme for the ability of this complex to perform multi-step reactions is presented. Despite the central role played by DPSL, cells maintained a lower level of protection after disruption of the dpsl gene, indicating a level of redundancy in the oxidative stress pathways of S. solfataricus. This work provides the first “omics” scale assessment of the oxidative stress response for an archeal organism and together with a network analysis using data from previous studies on bacteria and eukaryotes reveals evolutionarily conserved pathways where complex and overlapping defense mechanisms protect against oxygen toxicity.Item H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle(2018-01) Berry, Luke; Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R.; Nguyen, Diep M. N.; Schut, Gerrit J.; Adams, Michael W. W.; Peters, John W.; Boyd, Eric S.; Bothner, BrianRecent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP(+) oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron-sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units.Item Multi-level Omics Analysis Provides Insight to the Ignicoccus hospitalis-Nanoarchaeum equitans Association(2017-09) Rawle, Rachel A.; Hamerly, Timothy; Tripet, Brian P.; Giannone, Richard J.; Wurch, Louie; Hettich, Robert L.; Podar, Mircea; Copie, Valerie; Bothner, BrianBACKGROUND Studies of interspecies interactions are inherently difficult due to the complex mechanisms which enable these relationships. A model system for studying interspecies interactions is the marine hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans. Recent independently-conducted 'omics' analyses have generated insights into the molecular factors modulating this association. However, significant questions remain about the nature of the interactions between these archaea. METHODS We jointly analyzed multiple levels of omics datasets obtained from published, independent transcriptomics, proteomics, and metabolomics analyses. DAVID identified functionally-related groups enriched when I. hospitalis is grown alone or in co-culture with N. equitans. Enriched molecular pathways were subsequently visualized using interaction maps generated using STRING. RESULTS Key findings of our multi-level omics analysis indicated that I. hospitalis provides precursors to N. equitans for energy metabolism. Analysis indicated an overall reduction in diversity of metabolic precursors in the I. hospitalis-N. equitans co-culture, which has been connected to the differential use of ribosomal subunits and was previously unnoticed. We also identified differences in precursors linked to amino acid metabolism, NADH metabolism, and carbon fixation, providing new insights into the metabolic adaptions of I. hospitalis enabling the growth of N. equitans. CONCLUSIONS This multi-omics analysis builds upon previously identified cellular patterns while offering new insights into mechanisms that enable the I. hospitalis-N. equitans association. GENERAL SIGNIFICANCE Our study applies statistical and visualization techniques to a mixed-source omics data set to yield a more global insight into a complex system, that was not readily discernable from separate omics studies.Item Two functionally distinct NADP(+)-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus(2017-07) Nguyen, Diep M. N.; Schut, Gerrit J.; Zadvornyy, Oleg A.; Tokmina-Lukaszewska, Monika; Poudel, Saroj; Lipscomb, Gina L.; Adams, Leslie A.; Dinsmore, Jessica T.; Nixon, William J.; Boyd, Eric S.; Bothner, Brian; Peters, John W.; Adams, Michael W. W.Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP+ oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes and NfnII does not catalyze the NfnI bifurcating reaction. Instead it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and to also catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown.Item Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum(2013-08) Valenzuela, Jacob J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.; Bothner, Brian; Fields, Matthew W.Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.Item New technologies for studying biofilms(2015-08) Franklin, Michael J.; Chang, Connie B.; Akiyama, Tatsuya; Bothner, BrianThe results of recent biofilm characterizations have helped reveal the complexities of these surface-associated communities of microorganisms. The activities of the cells and the structure of the extracellular matrix material demonstrate that biofilm bacteria engage in a variety of physiological behaviors that are distinct from planktonic cells (1 – 3 ). For example, bacteria in biofilms are adapted to growth on surfaces, and most produce adhesins and extracellular polymers that allow the cells to firmly adhere to the surfaces or to neighboring cells ( 4 – 6 ). The extracellular material of biofilms contains polysaccharides, proteins, and DNA that form a glue-like substance for adhesion to the surface and for the three-dimensional (3D) biofilm architecture ( 4 ). The matrix material, although produced by the individual cells, forms structures that provide benefits for the entire community, including protection of the cells from various environmental stresses ( 7 – 9 ). Biofilm cells form a community and engage in intercellular signaling activities ( 10 – 19 ). Diffusible signaling molecules and metabolites provide cues for expression of genes that may benefit the entire community, such as genes for production of extracellular enzymes that allow the biofilm bacteria to utilize complex nutrient sources ( 18 , 20 – 22 ). Biofilm cells are not static. Many microorganisms have adapted to surface-associated motility, such as twitching and swarming motility ( 23 – 28 ). Cellular activities, including matrix production, intercellular signaling, and surface-associated swarming motility suggest that biofilms engage in communal activities. As a result, biofilms have been compared to multicellular organs where cells differentiate with specialized functions ( 2 , 29 ). However, bacteria do not always cooperate with each other. Biofilms are also sites of intense competition. The bacteria within biofilms compete for nutrients and space by producing toxic chemicals to inhibit or kill neighboring cells or inject toxins directly into neighboring cells through type VI secretion ( 30 – 33 ). Therefore, biofilm cells exhibit both communal and competitive activities.Item Unification of [FeFe]-hydrogenases into Three Structural and Functional Groups(2016-09) Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R.; Refai, Mohammed Y.; Schut, Gerrit J.; King, Paul W.; Maness, Pin-Ching; Adams, Michael W. W.; Peters, John W.; Bothner, Brian; Boyd, Eric S.Background: [FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2. Methods: To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods. Results: HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggest that they are post-translationally modified by phosphorylation. Conclusions: These results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA. General significance: This classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes.Item A periplasmic arsenite-binding protein involved in regulating arsenite oxidation(2011-12) Liu, Guanghui; Liu, Mengyao; Kim, Eun-Hae; Maaty, Walid S.; Bothner, Brian; Lei, Benfang; Rensing, Christopher; Wang, Gejiao; McDermott, Timothy R.Arsenic (As) is the most common toxic element in the environment, ranking first on the Superfund List of Hazardous Substances. Microbial redox transformations are the principal drivers of As chemical speciation, which in turn dictates As mobility and toxicity. Consequently, in order to manage or remediate environmental As, land managers need to understand how and why microorganisms react to As. Studies have demonstrated a two-component signal transduction system comprised of AioS (sensor kinase) and AioR (response regulator) is involved in regulating microbial AsIII oxidation, with the AsIII oxidase structural genes aioB and aioA being upregulated by AsIII. However, it is not known whether AsIII is first detected directly by AioS or by an intermediate. Herein we demonstrate the essential role of a periplasmic AsIII-binding protein encoded by aioX, which is upregulated by AsIII. An ΔaioX mutant is defective for upregulation of the aioBA genes and consequently AsIII oxidation. Purified AioX expressed without its TAT-type signal peptide behaves as a monomer (MW 32 kDa), and Western blots show AioX to be exclusively associated with the cytoplasmic membrane. AioX binds AsIII with a KD of 2.4 µM AsIII; however, mutating a conserved Cys108 to either alanine or serine resulted in lack of AsIII binding, lack of aioBA induction, and correlated with a negative AsIII oxidation phenotype. The discovery and characterization of AioX illustrates a novel AsIII sensing mechanism that appears to be used in a range of bacteria and also provides one of the first examples of a bacterial signal anchor protein.