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

Modulation of Gr1low monocyte subset impacts insulin sensitivity and weight gain upon high-fat diet in female mice

International audienceBackground/Objectives: Blood monocytes are expanded during obesity. However, the differential contribution of monocyte subsets in obesity-related metabolic disorders remains unknown. The aim of the study was to define the role of the Gr1low monocyte subset upon high-fat diet (HFD).Methods: We used transgenic female mouse models allowing the modulation of circulating Gr1low monocyte number (decreased number in CX3CR1−/− mice and increased number in CD11c-hBcl2 mice) and studied obesity upon HFD.Results: We reported here that HFD induced monocytosis in mice, preferentially due to Gr1low monocyte expansion, and was associated with a specific upregulation of CD11c on that subset. Using mice models with altered Gr1low monocyte number, we found a striking correlation between Gr1low monocytes, bodyweight (BW) and insulin resistance (RT) status. Indeed, CX3CR1−/− female mice, with reduced Gr1low monocytes upon HFD, showed increased RT and a pro-inflammatory profile of the adipose tissue (AT) despite a lower BW. Conversely, mice expressing the anti-apoptotic gene hBcl2 in CD11c-expressing cells have increased Gr1low monocytes, higher insulin sensitivity upon HFD and an anti-inflammatory profile of the AT. Finally, increasing Gr1low monocytes in Gr1low-defective CX3CR1−/− mice rescued BW loss in these mice.Conclusions: By using transgenic female mice and adoptive transfer experiments, we established the evidence for a correlation between Gr1low monocyte subset and weight gain and RT. Hence, this specific Gr1low monocyte subset could be used as a target for acting on AT inflammation and RT