Browsing by Author "Eilers, Brian J."

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Copper modulates sex-specific fructose hepatoxicity in nonalcoholic fatty liver disease (NALFD) Wistar rat models
(2020-04) Morrell, Austin; Tripet, Brian P.; Eilers, Brian J.; Tegman, Megan; Thompson, Damon; Copie, Valerie; Burkhead, Jason L.
This study aimed to characterize the impact of dietary copper on the biochemical and hepatic metabolite changes associated with fructose toxicity in a Wistar rat model of fructose-induced liver disease. Twenty-four male and 24 female, 6-week-old, Wister rats were separated into four experimental dietary treatment groups (6 males and 6 females per group), as follows: (1) a control diet: containing no fructose with adequate copper (i.e., CuA/0% Fruct); (2) a diet regimen identical to the control and supplemented with 30% w/v fructose in the animals' drinking water (CuA/30% Fruct); (3) a diet identical to the control diet but deficient in copper content (CuD/0% Fruct) and (4) a diet identical to the control diet but deficient in copper content and supplemented with 30% w/v fructose in the drinking water (CuD/30% Fruct). The animals were fed the four diet regimens for 5 weeks, followed by euthanization and assessment of histology, elemental profiles and identification and quantitation of liver metabolites. Results from 1H nuclear magnetic resonance metabolomics revealed mechanistic insights into copper modulation of fructose hepatotoxicity through identification of distinct metabolic phenotypes that were highly correlated with diet and sex. This study also identified previously unknown sex-specific responses to both fructose supplementation and restricted copper intake, while the presence of adequate dietary copper promoted most pronounced fructose-induced metabolite changes.
Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis
(2020-02) Steward, Katherine F.; Eilers, Brian J.; Tripet, Brian P.; Fuchs, Amanda; Dorle, Michael; Rawle, Rachel A.; Soriano, Berliza; Balasubramanian, Narayanaganesh; Copie, Valerie; Bothner, Brian; Hatzenpichler, Roland
BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG); L-methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools.
Solution Structure and Molecular Determinants of Hemoglobin Binding of the first NEAT Domain of IsdB in Staphylococcus aureus
(2014-06) Fonner, Brittany A.; Tripet, Brian P.; Eilers, Brian J.; Stanisich, Jessica J.; Sullivan-Springhetti, Rose K.; Moore, Rebecca; Liu, Mengyao; Lei, Benfang; Copie, Valerie
The human pathogen Staphylococcus aureus acquires heme iron from hemoglobin (Hb) via the action of a series of iron-regulated surface determinant (Isd) proteins. The cell wall anchored IsdB protein is recognized as the predominant Hb receptor, and is comprised of two NEAr transporter (NEAT) domains that act in concert to bind, extract, and transfer heme from Hb to downstream Isd proteins. Structural details of the NEAT 2 domain of IsdB have been investigated, but the molecular coordination between NEAT 2 and NEAT 1 to extract heme from hemoglobin has yet to be characterized. To obtain a more complete understanding of IsdB structure and function, we have solved the 3D solution structure of the NEAT 1 domain of IsdB (IsdBN1) spanning residues 125–272 of the full-length protein by NMR. The structure reveals a canonical NEAT domain fold and has particular structural similarity to the NEAT 1 and NEAT 2 domains of IsdH, which also interact with Hb. IsdBN1 is also comprised of a short N-terminal helix, which has not been previously observed in other NEAT domain structures. Interestingly, the Hb binding region (loop 2 of IsdBN1) is disordered in solution. Analysis of Hb binding demonstrates that IsdBN1 can bind metHb weakly and the affinity of this interaction is further increased by the presence of IsdB linker domain. IsdBN1 loop 2 variants reveal that phenylalanine 164 (F164) of IsdB is necessary for Hb binding and rapid heme transfer from metHb to IsdB. Together, these findings provide a structural role for IsdBN1 in enhancing the rate of extraction of metHb heme by the IsdB NEAT 2 domain.
Structural Basis for the Mechanism of ATP-Dependent Acetone Carboxylation
(2017-08) Mus, Florence; Eilers, Brian J.; Alleman, Alexander B.; Kabasakal, Burak V.; Wells, Jennifer N.; Murray, James W.; Nocek, Boguslaw P.; DuBois, Jennifer L.; Peters, John W.
Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon dioxide gas (CO2) or its hydrated form, bicarbonate (HCO3 −), into target molecules. Acetone carboxylases (ACs) catalyze the conversion of substrates acetone and HCO3 − to form the product acetoacetate. Many bicarbonate-incorporating carboxylases rely on the organic cofactor biotin for the activation of bicarbonate. ACs contain metal ions but not organic cofactors, and use ATP to activate substrates through phosphorylation. How the enzyme coordinates these phosphorylation events and new C-C bond formation in the absence of biotin has remained a mystery since these enzymes were discovered. The first structural rationale for acetone carboxylation is presented here, focusing on the 360 kDa (αβγ)2 heterohexameric AC from Xanthobacter autotrophicus in the ligand-free, AMP-bound, and acetate coordinated states. These structures suggest successive steps in a catalytic cycle revealing that AC undergoes large conformational changes coupled to substrate activation by ATP to perform C-C bond ligation at a distant Mn center. These results illustrate a new chemical strategy for the conversion of CO2 into biomass, a process of great significance to the global carbon cycle.