Characterization of 4-HNE Modified L-FABP Reveals Alterations in Structural and Functional Dynamics

4-Hydroxynonenal (4-HNE) is a reactive α,β-unsaturated aldehyde produced during oxidative stress and subsequent lipid peroxidation of polyunsaturated fatty acids. The reactivity of 4-HNE towards DNA and nucleophilic amino acids has been well established. In this report, using proteomic approaches, liver fatty acid-binding protein (L-FABP) is identified as a target for modification by 4-HNE. This lipid binding protein mediates the uptake and trafficking of hydrophobic ligands throughout cellular compartments. Ethanol caused a significant decrease in L-FABP protein (P<0.001) and mRNA (P<0.05), as well as increased poly-ubiquitinated L-FABP (P<0.001). Sites of 4-HNE adduction on mouse recombinant L-FABP were mapped using MALDI-TOF/TOF mass spectrometry on apo (Lys57 and Cys69) and holo (Lys6, Lys31, His43, Lys46, Lys57 and Cys69) L-FABP. The impact of 4-HNE adduction was found to occur in a concentration-dependent manner; affinity for the fluorescent ligand, anilinonaphthalene-8-sulfonic acid, was reduced from 0.347 µM to Kd1 = 0.395 µM and Kd2 = 34.20 µM. Saturation analyses revealed that capacity for ligand is reduced by approximately 50% when adducted by 4-HNE. Thermal stability curves of apo L-FABP was also found to be significantly affected by 4-HNE adduction (ΔTm = 5.44°C, P<0.01). Computational-based molecular modeling simulations of adducted protein revealed minor conformational changes in global protein structure of apo and holo L-FABP while more apparent differences were observed within the internal binding pocket, revealing reduced area and structural integrity. New solvent accessible portals on the periphery of the protein were observed following 4-HNE modification in both the apo and holo state, suggesting an adaptive response to carbonylation. The results from this study detail the dynamic process associated with L-FABP modification by 4-HNE and provide insight as to how alterations in structural integrity and ligand binding may a contributing factor in the pathogenesis of ALD

Investigation of the obscuring circumnuclear torus in the active galaxy Mrk231

Active galaxies are characterized by prominent emission from their nuclei. In the 'unified' view of active galaxies, the accretion of material onto a massive compact object-now generally believed to be a black hole-provides the fundamental power source(1). Obscuring material along the line of sight can account for the observed differences in the nuclear emission(2,3), which determine the classification of AGN (for example, as Seyfert 1 or Seyfert 2 galaxies). Although the physical processes of accretion have been confirmed observationally(4,5), the structure and extent of the obscuring material have not been determined. Here we report observations of powerful hydroxyl (OH) line emissions that trace this obscuring material within the circumnuclear environment of the galaxy Markarian 231. The hydroxyl (mega)-maser emission shows the characteristics of a rotating, dusty, molecular torus (or thick disk) located between 30 and 100 pc from the central engine. We now have a clear view of the physical conditions, the kinematics and the spatial structure of this material on intermediate size scales, confirming the main tenets of unification models