Novel ligands that target the mitochondrial membrane protein mitoNEET.

Ligands of the thiazolidinedione (TZD) class of compounds, pioglitazone (Actos™) and rosiglitazone (Avandia™) are currently approved for treatment of type 2 diabetes and are known to bind to the PPAR-γ nuclear receptor subtype. Recent evidence suggesting PPAR-γ independent action of the TZDs led to the discovery of a novel integral outer mitochondrial membrane protein, mitoNEET. In spite of the several reported X-ray crystal structures of the unbound form of mitoNEET, the location and nature of the mitoNEET ligand binding sites (LBS) remain unknown. In this study, a molecular blind docking (BD) method was used to discover potential mitoNEET LBS and novel ligands, utilizing the program AutoDock Vina (v 1.0.2). Validation of BD was performed on the PPAR-γ receptor (PDB ID: 1ZGY) with the test compound rosiglitazone, demonstrating that the binding conformation of rosiglitazone determined by AutoDock Vina matches well with that of the cocrystallized ligand (root mean square deviation of the heavy atoms 1.45Å). The locations and a general ligand binding interaction model for the LBS were determined, leading to the discovery of novel mitoNEET ligands. An in vitro fluorescence binding assay utilizing purified recombinant mitoNEET protein was used to determine the binding affinity of a predicted mitoNEET ligand, and the data obtained is in good agreement with AutoDock Vina results. The discovery of potential mitoNEET ligand binding sites and novel ligands, opens up the possibility for detailed structural studies of mitoNEET-ligand complexes, as well as rational design of novel ligands specifically targeted for mitoNEET.

In situ metabolic flux analysis to quantify the liver metabolic response to experimental burn injury.

Trauma such as burns induces a hypermetabolic response associated with altered central carbon and nitrogen metabolism. The liver plays a key role in these metabolic changes; however, studies to date have evaluated the metabolic state of liver using ex vivo perfusions or isotope labeling techniques targeted to specific pathways. Herein, we developed a unique mass balance approach to characterize the metabolic state of the liver in situ, and used it to quantify the metabolic changes to experimental burn injury in rats. Rats received a sham (control uninjured), 20% or 40% total body surface area (TBSA) scald burn, and were allowed to develop a hypermetabolic response. One day prior to evaluation, all animals were fasted to deplete glycogen stores. Four days post-burn, blood flow rates in major vessels of the liver were measured, and blood samples harvested. We combined measurements of metabolite concentrations and flow rates in the major vessels entering and leaving the liver with a steady-state mass balance model to generate a quantitative picture of the metabolic state of liver. The main findings were: (1) Sham-burned animals exhibited a gluconeogenic pattern, consistent with the fasted state; (2) the 20% TBSA burn inhibited gluconeogenesis and exhibited glycolytic-like features with very few other significant changes; (3) the 40% TBSA burn, by contrast, further enhanced gluconeogenesis and also increased amino acid extraction, urea cycle reactions, and several reactions involved in oxidative phosphorylation. These results suggest that increasing the severity of injury does not lead to a simple dose-dependent metabolic response, but rather leads to qualitatively different responses.

Interactions among alpha-synuclein, dopamine, and biomembranes: some clues for understanding neurodegeneration in Parkinson's disease.

Parkinson's disease (PD) is a neurologic disorder resulting from the loss of dopaminergic neurons in the brain. Two lines of evidence suggest that the protein alpha-synuclein plays a role in the pathogenesis of PD: Fibrillar alpha-synuclein is a major component of Lewy bodies in diseased neurons, and two mutations in alpha-synuclein are linked to early-onset disease. Accordingly, the fibrillization of alpha-synuclein is proposed to contribute to neurodegeneration in PD. In this report, we provide evidence that oligomeric intermediates of the alpha-synuclein fibrillization pathway, termed protofibrils, might be neurotoxic. Analyses of protofibrillar alpha-synuclein by atomic force microscopy and electron microscopy indicate that the oligomers consist of spheres, chains, and rings. alpha-Synuclein protofibrils permeabilize synthetic vesicles and form pore-like assemblies on the surface of brain-derived vesicles. Dopamine reacts with alpha-synuclein to form a covalent adduct that slows the conversion of protofibrils to fibrils. This finding suggests that cytosolic dopamine in dopaminergic neurons promotes the accumulation of toxic alpha-synuclein protofibrils, which might explain why these neurons are most vulnerable to degeneration in PD. Finally, we note that aggregation of alpha-synuclein likely occurs via different mechanisms in the cell versus the test tube. For example, the binding of alpha-synuclein to cellular membranes might influence its self-assembly. To address this point, we have developed a yeast model that might enable the selection of random alpha-synuclein mutants with different membrane-binding affinities. These variants might be useful to test whether membrane binding by alpha-synuclein is necessary for neurodegeneration in transgenic animal models of PD.