Biochemical Control of the Mitochondrial Protein MitoNEET by Biological Thiols and Lipid-derived Electrophiles.

MitoNEET is a mitochondrial [2Fe-2S] protein known for its involvement in cellular metabolism, iron regulation, and oxidative stress. The protein has been associated with diseases ranging from diabetes to Parkinson's disease which has prompted development of compounds designed to selectively target mitoNEET. Unfortunately, drug development is limited due to a lack of understanding on the mechanistic level how mitoNEET integrates into pathophysiological processes. In particular, biological compounds that govern mitoNEET function are still ill defined. We demonstrate an oxygen-dependent reaction with biological thiols catalyzed by mitoNEET. Furthermore, we observed that formation of a covalently linked mitoNEET homodimer is controlled by both thiols and lipid-derived electrophiles. Finally, we demonstrate that reduced glutathione (L-GSH) regulates the reactivity of two lipid-derived biomarkers of oxidative stress, 4-HNE and 4-ONE, towards mitoNEET. We find that exposure to L-GSH prior to treatment with either of the electrophilic aldehydes prevents the formation of the covalently linked mitoNEET dimer. Meanwhile, addition of L-GSH after electrophile treatment recovers mitoNEET from the 4-HNE induced modification but not from the modification induced by 4-ONE. Our results collectively suggest that the thiol-electrophile redox balance governing ferroptotic cell death also controls mitoNEET's state at multiple biochemical levels. These results indicate a possible role for mitoNEET in thiol-mediated oxidative stress and may inform about development of probes designed to modulate mitoNEET activity to improve pathophysiological states.

Global changes to HepG2 cell metabolism in response to galactose treatment.

Tumor cell proliferation requires sufficient metabolic flux through the pentose phosphate pathway to meet the demand for biosynthetic precursors and to increase protection against oxidative stress which in turn requires an upregulation of substrate flow through glycolysis. This metabolic poise is often coupled with a shift in ATP production from mitochondrial OXPHOS to substrate-level phosphorylation. Despite major advances that were facilitated by using tumor-derived cell lines in research areas spanning from membrane to cytoskeletal biology, this distorted metabolic profile limits their impact as a model in physiology and toxicology. Substitution of glucose with galactose in the cell culture medium has been demonstrated to shift ATP production from substrate-level phosphorylation to mitochondrial OXPHOS. This increase in oxygen utilization is coupled to a global metabolic reorganization with potential impacts on macromolecule biosynthesis and cellular redox homeostasis, but a comprehensive analysis on the effects of sugar substitution in tumor-derived cells is still missing. To address this gap in knowledge we performed transcriptomic and metabolomic analyses on human hepatocellular carcinoma (HepG2) cells adapted to either glucose or galactose as the aldohexose source. We observed a shift toward oxidative metabolism in all primary metabolic pathways at both transcriptomic and metabolomic levels. We also observed a decrease in nicotinamide dinucleotide (NAD(P)) levels and subcellular NAD+-to-NADH ratios in cells cultured with galactose compared with glucose control cells. Our results suggest that galactose reduces both glycolytic and biosynthetic flux and restores a metabolic poise in HepG2 cells that closely reflects the metabolic state observed in primary hepatocytes.

Tradeoffs of warm adaptation in aquatic ectotherms: Live fast, die young?

In the face of a changing climate, questions regarding sub-lethal effects of elevated habitat temperature on the physiology of ectotherms remain unanswered. In particular, long-term responses of ectotherms to the warming trend in tropical regions are unknown, and understudied due to the difficulties in specimen and community traceability. In freshwater lakes employed as cooling reservoirs for power plants, increased physiological stress from high water temperature can potentially alter the community structure of fishes. We employ this highly tractable system to assess how thermal regimes can alter the physiology and ecology of aquatic species. We documented a significantly reduced lifespan, growth performance, and a shift in the age structure towards younger individuals in the thermally- impacted Coffeen Lake population of bluegill (Lepomis macrochirus), compared to a non-impacted control group (Lake Mattoon). Average age calculated for the Lake Mattoon population was 2.42 years, whereas the average age of bluegill from Coffeen Lake was only 0.96 years, and average specimen mass in Lake Mattoon was more than six times that of Coffeen Lake. During laboratory cross-acclimation studies of bluegill from Lake Mattoon at 17.5 and 35.0°C, citrate synthase (CS) activity obtained from white muscle was regulated through acclimation, whereas cold-acclimated specimens exhibited twice the activity at 25°C, if compared to CS activity values from warm-acclimated specimens. This study raises the questions about the causal relationships between physiological performance and habitat temperature, in particular how thresholds in an organism's physiology may modulate their community structure, and consequently their ecological success.

Binding of urate and caffeine to hemocyanin of the lobster Homarus vulgaris (E.) as studied by isothermal titration calorimetry.

Hemocyanin serves as an oxygen carrier in the hemolymph of the European lobster Homarus vulgaris. The oxygen binding behavior of the pigment is modulated by metabolic effectors such as lactate and urate. Urate and caffeine binding to 12-meric hemocyanin (H. vulgaris) was studied using isothermal titration calorimetry (ITC). Binding isotherms were determined for fully oxygenated hemocyanin between pH 7.55 and 8.15. No pH dependence of the binding parameters could be found for either effector. Since the magnitude of the Bohr effect depends on the urate concentration, the absence of any pH dependence of urate and caffeine binding to oxygenated hemocyanin suggests two conformations of the pigment under deoxygenated conditions. Urate binds to two identical binding sites (n = 2) each with a microscopic binding constant K of 8500 M(-1) and an enthalpy change DeltaH degrees of -32.3 kcal mol(-1). Caffeine binds cooperatively to hemocyanin with two microscopic binding constants: K(1) = 14 100 M(-1) and K(2) = 40 400 M(-1). The corresponding enthalpy changes in binding are as follows: DeltaH degrees (1) = -23.3 kcal mol(-1) and DeltaH degrees (2) = -27.1 kcal mol(-1). The comparison of urate and caffeine binding to the oxygenated pigment indicates the existence of two protein conformations for oxygen-saturated hemocyanin. Since effector binding is not influenced by protons, four different conformations are required to create a convincing explanation for caffeine and urate binding curves. This was predicted earlier on the basis of the analysis of oxygen binding to lobster hemocyanin, employing the nesting model.