Benjamin J. Stewart

Overview of lipid peroxidation products and hepatic protein modification in alcoholic liver disease

OBJECTIVES Oxidative stress is one component of alcoholic liver disease (ALD) that is manifested in the peroxidation of cellular lipids producing the electrophile, 4-hydroxynonenal (4-HNE). This electrophile is proposed to modify essential cellular proteins resulting in loss of protein function and cellular homeostasis. Studies were initiated to identify hepatic proteins that are targets of 4-HNE modification and determine their relationship with progression of the early stages of ALD. METHODS Rat and mouse models were developed using the Lieber-DeCarli diet to simulate early stages of ALD consisting of fatty liver (steatosis) and hepatocellular injury indicated by a 1.5-2-fold elevation of plasma ALT activity. Liver samples obtained from control and ethanol treated animals were subjected to two-dimensional electrophoresis and immunoblotting using polyclonal antibodies generated against 4-HNE epitopes for detection of proteins modified by 4-HNE. Following identification of 4-HNE adducted proteins, the respective recombinant proteins modified with physiologic concentrations of 4-HNE were evaluated to determine the functional consequences of 4-HNE modification. RESULTS One group of proteins identified included Hsp70, Hsp90 and protein disulfide isomerase (PDI), all of which are involved in protein folding or processing are targets of adduction. In vitro assays indicated significant impairment of the protein activities following modification with physiologically relevant concentrations of 4-HNE. Liver fatty acid binding protein, L-FABP, was also identified as a target and additional studies revealed that the levels of this protein were significantly decreased because of chronic ethanol ingestion. Erk1/2 was identified as a target for modification and subsequently determined to have impaired activity. CONCLUSIONS Inhibition of Hsp70, Hsp90 and PDI function could be involved in initiation of the early phases of ER stress contributing to stimulation and accumulation of hepatic lipids. Likewise, impairment of L-FABP activity could also disrupt lipid transport also contributing to steatosis. The modification and inhibition of Erk1/2 by 4-HNE may also contribute to the decreased hepatocellular proliferation associated with ALD. Collectively, these results provide new information concerning the mechanisms whereby the modification of hepatic proteins by 4-HNE contributes to ALD.

Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae†

Nicotinamide adenine dinucleotide (NAD+) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD+ decomposition products. NAD+ biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD+ biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD+ and NADH (the reduced form of NAD+) analyses on BY4742 wild‐type, NAD+ salvage pathway knockout (npt1Δ) and NAD+ de novo pathway knockout (qpt1Δ) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized 14C labelled nicotinic acid in the culture media combined with HPLC speciation and both UV and 14C detection to quantitate the total amounts of NAD+ and NADH and the amounts derived from the salvage pathway. We observed that wild‐type and qpt1Δ yeast exclusively utilized extracellular nicotinic acid for NAD+ and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions, suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observed that NAD+ concentrations decreased in all three strains under CR. However, unlike the wild‐type strain, NADH concentrations did not decrease and NAD+: NADH ratios did not increase under CR for either knockout strain. Lifespan analyses revealed that CR resulted in a lifespan increase of approximately 25% for the wild‐type and qpt1Δ strains, while no increase in lifespan was observed for the npt1Δ strain. In combination, these data suggest that having a functional salvage pathway is required for lifespan extension under CR. Copyright

Directly coupled high-performance liquid chromatography-accelerator mass spectrometry measurement of chemically modified protein and peptides

Quantitation of low-abundance protein modifications involves significant analytical challenges, especially in biologically important applications, such as studying the role of post-translational modifications in biology and measurement of the effects of reactive drug metabolites. (14)C labeling combined with accelerator mass spectrometry (AMS) provides exquisite sensitivity for such experiments. Here, we demonstrate real-time (14)C quantitation of high-performance liquid chromatography (HPLC) separations by liquid sample accelerator mass spectrometry (LS-AMS). By enabling direct HPLC-AMS coupling, LS-AMS overcomes several major limitations of conventional HPLC-AMS, where individual HPLC fractions must be collected and converted to graphite before measurement. To demonstrate LS-AMS and compare the new technology to traditional solid sample AMS (SS-AMS), reduced and native bovine serum albumin (BSA) was modified by (14)C-iodoacetamide, with and without glutathione present, producing adducts on the order of 1 modification in every 10(6) to 10(8) proteins. (14)C incorporated into modified BSA was measured by solid carbon AMS and LS-AMS. BSA peptides were generated by tryptic digestion. Analysis of HPLC-separated peptides was performed in parallel by LS-AMS, fraction collection combined with SS-AMS, and (for peptide identification) electrospray ionization and tandem mass spectrometry (ESI-MS/MS). LS-AMS enabled (14)C quantitation from ng sample sizes and was 100 times more sensitive to (14)C incorporated in HPLC-separated peptides than SS-AMS, resulting in a lower limit of quantitation of 50 zmol (14)C/peak. Additionally, LS-AMS turnaround times were minutes instead of days, and HPLC trace analyses required 1/6th the AMS instrument time required for analysis of graphite fractions by SS-AMS.

Inhibiting tryptophan metabolism enhances interferon therapy in kidney cancer

Renal cell carcinoma (RCC) is increasing in incidence, and a complete cure remains elusive. While immune-checkpoint antibodies are promising, interferon-based immunotherapy has been disappointing. Tryptophan metabolism, which produces immunosuppressive metabolites, is enhanced in RCC. Here we show indolamine-2,3-dioxygenase-1 (IDO1) expression, a kynurenine pathway enzyme, is increased not only in tumor cells but also in the microenvironment of human RCC compared to normal kidney tissues. Neither kynurenine metabolites nor IDO inhibitors affected the survival or proliferation of human RCC or murine renal cell adenocarcinoma (RENCA) cells in vitro. However, interferon-gamma (IFNγ) induced high levels of IDO1 in both RCC and RENCA cells, concomitant with enhanced kynurenine levels in conditioned media. Induction of IDO1 by IFNα was weaker than by IFNγ. Neither the IDO1 inhibitor methyl-thiohydantoin-DL-tryptophan (MTH-trp) nor IFNα alone inhibited RENCA tumor growth, however the combination of MTH-trp and IFNα reduced tumor growth compared to IFNα. Thus, the failure of IFNα therapy for human RCC is likely due to its inability to overcome the immunosuppressive environment created by increased IDO1. Based on our data, and given that IDO inhibitors are already in clinical trials for other malignancies, IFNα therapy with an IDO inhibitor should be revisited for RCC.

D-Lactate production as a function of glucose metabolism in Saccharomyces cerevisiae

Methylglyoxal, a reactive, toxic dicarbonyl, is generated by the spontaneous degradation of glycolytic intermediates. Methylglyoxal can form covalent adducts with cellular macromolecules, potentially disrupting cellular function. We performed experiments using the model organism Saccharomyces cerevisiae, grown in media containing low, moderate and high glucose concentrations, to determine the relationship between glucose consumption and methylglyoxal metabolism. Normal growth experiments and glutathione depletion experiments showed that metabolism of methylglyoxal by log‐phase yeast cultured aerobically occurred primarily through the glyoxalase pathway. Growth in high‐glucose media resulted in increased generation of the methylglyoxal metabolite d‐lactate and overall lower efficiency of glucose utilization as measured by growth rates. Cells grown in high‐glucose media maintained higher glucose uptake flux than cells grown in moderate‐glucose or low‐glucose media. Computational modelling showed that increased glucose consumption may impair catabolism of triose phosphates as a result of an altered NAD+:NADH ratio. Copyright

YEAST DYNAMIC METABOLIC FLUX MEASUREMENT IN NUTRIENT-RICH MEDIA BY HPLC AND ACCELERATOR MASS SPECTROMETRY

Metabolic flux, the flow of metabolites through networks of enzymes, represents the dynamic productive output of cells. Improved understanding of intracellular metabolic fluxes will enable targeted manipulation of metabolic pathways of medical and industrial importance to a greater degree than is currently possible. Flux balance analysis (FBA) is a constraint-based approach to modeling metabolic fluxes, but its utility is limited by a lack of experimental measurements. Incorporation of experimentally measured fluxes as system constraints will significantly improve the overall accuracy of FBA. We applied a novel, two-tiered approach in the yeast Saccharomyces cerevisiae to measure nutrient consumption rates (extracellular fluxes) and a targeted intracellular flux using a (14)C-labeled precursor with HPLC separation and flux quantitation by accelerator mass spectrometry (AMS). The use of AMS to trace the intracellular fate of (14)C-glutamine allowed the calculation of intracellular metabolic flux through this pathway, with glutathione as the metabolic end point. Measured flux values provided global constraints for the yeast FBA model which reduced model uncertainty by more than 20%, proving the importance of additional constraints in improving the accuracy of model predictions and demonstrating the use of AMS to measure intracellular metabolic fluxes. Our results highlight the need to use intracellular fluxes to constrain the models. We show that inclusion of just one such measurement alone can reduce the average variability of model predicted fluxes by 10%.

Temporal succession in carbon incorporation from macromolecules by particle-attached bacteria in marine microcosms.

We investigated bacterial carbon assimilation from stable isotope-labelled macromolecular substrates (proteins; lipids; and two types of polysaccharides, starch and cellobiose) while attached to killed diatom detrital particles during laboratory microcosms incubated for 17 days. Using Chip-SIP (secondary ion mass spectrometry analysis of RNA microarrays), we identified generalist operational taxonomic units (OTUs) from the Gammaproteobacteria, belonging to the genera Colwellia, Glaciecola, Pseudoalteromonas and Rheinheimera, and from the Bacteroidetes, genera Owenweeksia and Maribacter, that incorporated the four tested substrates throughout the incubation period. Many of these OTUs exhibited the highest isotope incorporation relative to the others, indicating that they were likely the most active. Additional OTUs from the Gammaproteobacteria, Bacteroidetes and Alphaproteobacteria exhibited generally (but not always) lower activity and did not incorporate all tested substrates at all times, showing species succession in organic carbon incorporation. We also found evidence to suggest that both generalist and specialist OTUs changed their relative substrate incorporation over time, presumably in response to changing substrate availability as the particles aged. This pattern was demonstrated by temporal succession from relatively higher starch incorporation early in the incubations, eventually switching to higher cellobiose incorporation after 2 weeks.

Quantitative In Silico analysis of transient metabolism of acetaminophen and associated causes of hepatotoxicity in humans

PurposeAlthough safe at therapeutic levels, excess intake of acetaminophen can lead to hepatic injury or acute liver failure (ALF). A number of different factors influence metabolism and hepatotoxicity of acetaminophen in patients. Three of the most important are a patient’s physiological response to fasting, alcohol consumption, and chronic acetaminophen consumption. The molecular and enzymatic underpinnings for these processes have been extensively studied. The purpose of this study is to examine and quantify the effects of the noted conditions, provide possible reasons for conflicting clinical observations, and examine dangers associated with uptake of therapeutic doses of acetaminophen.MethodsIn order to gain a better understanding of the transient hepatic changes associated with each physiological and nutritional process, examine risks of ALF associated with individuals based on their unique lifestyle and health issues, and predict improved dosing strategies, a multi-compartmented physiologically-based pharmacokinetic (PBPK) model of acetaminophen metabolism in adult humans was developed. By varying the parameters of this model, changes in metabolism of acetaminophen and its toxic byproducts for a variety of medically relevant conditions were assessed.ResultsSimulated results indicate that in case of chronic ingestion of acetaminophen, the increased rate of glucuronidation plays a significant role in protecting patients from liver damage following uptake of excessive quantities. Analysis of metabolism of acetaminophen in persons who have imbibed excessive amounts of alcohol show that the primary reason for hepatotoxicity in such individuals is decreased availability of glutathione in the liver and not the observed increased production of toxic byproducts. When the glutathione depleting effects of alcohol consumption are combined with those associated with chronic acetaminophen use, intake of slightly higher quantities than the recommended therapeutic doses of acetaminophen can result in initiation of hepatotoxicity.ConclusionsThe results of simulations show that, in healthy and well-fed individuals, chronic uptake of acetaminophen doses even five times the therapeutic recommendations should be safe. However, in persons who have diminished hepatic glutathione regeneration capacities, depending on the magnitude of this deleterious shortcoming, minor overdoses can result in hepatotoxicity. Hence, it can be concluded that for such persons, acetaminophen is just as toxic as any other compound that would generate reactive oxidative species.

Prospects for Fungal Bioremediation of Acidic Radioactive Waste Sites: Characterization and Genome Sequence of Rhodotorula taiwanensis MD1149

Highly concentrated radionuclide waste produced during the Cold War era is stored at US Department of Energy (DOE) production sites. This radioactive waste was often highly acidic and mixed with heavy metals, and has been leaking into the environment since the 1950s. Because of the danger and expense of cleanup of such radioactive sites by physicochemical processes, in situ bioremediation methods are being developed for cleanup of contaminated ground and groundwater. To date, the most developed microbial treatment proposed for high-level radioactive sites employs the radiation-resistant bacterium Deinococcus radiodurans. However, the use of Deinococcus spp. and other bacteria is limited by their sensitivity to low pH. We report the characterization of 27 diverse environmental yeasts for their resistance to ionizing radiation (chronic and acute), heavy metals, pH minima, temperature maxima and optima, and their ability to form biofilms. Remarkably, many yeasts are extremely resistant to ionizing radiation and heavy metals. They also excrete carboxylic acids and are exceptionally tolerant to low pH. A special focus is placed on Rhodotorula taiwanensis MD1149, which was the most resistant to acid and gamma radiation. MD1149 is capable of growing under 66 Gy/h at pH 2.3 and in the presence of high concentrations of mercury and chromium compounds, and forming biofilms under high-level chronic radiation and low pH. We present the whole genome sequence and annotation of R. taiwanensis strain MD1149, with a comparison to other Rhodotorula species. This survey elevates yeasts to the frontier of biologys most radiation-resistant representatives, presenting a strong rationale for a role of fungi in bioremediation of acidic radioactive waste sites.

High Initial Sputter Rate Found for Vaccinia Virions Using Isotopic Labeling, NanoSIMS, and AFM

High-lateral-resolution secondary ion mass spectrometry (SIMS) has the potential to provide functional and depth resolved information from small biological structures, such as viral particles (virions) and phage, but sputter rate and sensitivity are not characterized at shallow depths relevant to these structures. Here we combine stable isotope labeling of the DNA of vaccinia virions with correlated SIMS imaging depth profiling and atomic force microscopy (AFM) to develop a nonlinear, nonequilibrium sputter rate model for the virions and validate the model on the basis of reconstructing the location of the DNA within individual virions. Our experiments with a Cs+ beam show an unexpectedly high initial sputter rate (∼100 um2·nm·pA-1·s-1) with a rapid decline to an asymptotic rate of 0.7 um2·nm·pA-1·s-1 at an approximate depth of 70 nm. Correlated experiments were also conducted with glutaraldehyde-fixed virions, as well as O- and Ga+ beams, yielding similar results. Based on our Cs+ sputter rate model, the labeled DNA in the virion was between 50 and 90 nm depth in the virion core, consistent with expectations, supporting our conclusions. Virion densification was found to be a secondary effect. Accurate isotopic ratios were obtained from the initiation of sputtering, suggesting that isotopic tracers could be successfully used for smaller virions and phage.