Leukotrienes (LT) are lipid mediators of the inflammatory response that are linked to asthma and atherosclerosis. LT biosynthesis is initiated by 5-lipoxygenase (5-LOX) with the assistance of the substrate-binding 5-LOX-activating protein at the nuclear membrane. Here, we contrast the structural and functional consequences of the binding of two natural product inhibitors of 5-LOX. The redox-type inhibitor nordihydroguaiaretic acid (NDGA) is lodged in the 5-LOX active site, now fully exposed by disordering of the helix that caps it in the apo-enzyme. In contrast, the allosteric inhibitor 3-acetyl-11-keto-beta-boswellic acid (AKBA) from frankincense wedges between the membrane-binding and catalytic domains of 5-LOX, some 30 Å from the catalytic iron. While enzyme inhibition by NDGA is robust, AKBA promotes a shift in the regiospecificity, evident in human embryonic kidney 293 cells and in primary immune cells expressing 5-LOX.
The enzyme encoded by the ALOX15B gene has been linked to the development of atherosclerotic plaques in humans and in a mouse model of hypercholesterolemia. In vitro, these enzymes, which share 78% sequence identity, generate distinct products from their substrate arachidonic acid: the human enzyme, a 15-S-hydroperoxy product; and the murine enzyme, an 8-S-product. We probed the activities of these enzymes with nanodiscs as membrane mimics to determine whether they can access substrate esterified in a bilayer and characterized their activities at the membrane interface. We observed that both enzymes transform phospholipid-esterified arachidonic acid to a 15-S-product. Moreover, when expressed in transfected HEK cells, both enzymes result in significant increases in the amounts of 15-hydroxyderivatives of eicosanoids detected. In addition, we show that 15-LOX-2 is distributed at the plasma membrane when the HEK293 cells are stimulated by the addition Ca 2؉ ionophore and that cellular localization is dependent upon the presence of a putative membrane insertion loop. We also report that sequence differences between the human and mouse enzymes in this loop appear to confer distinct mechanisms of enzyme-membrane interaction for the homologues.A macrophage 2 activity has been linked to elevated levels of oxidized lipids through several experimental approaches that include the heterologous expression of human 15-LOX in a mouse model of hyperlipidemia (1) and pharmacological inhibition of 15-LOX activity (2, 3). These lipid oxidation products can enter the extracellular pool of cholesterol esters transported by LDL (4) and promote the pathological consequences of elevated LDL cholesterol levels (5). Macrophages that take up LDL laden with oxidized lipids are transformed to foam cells, an event that leads to further inflammation and apoptosis and results in the formation of atherosclerotic plaques. Recently, Magnusson et al. (6) demonstrated that silencing production of 15-LOX-2 (the product of the ALOX15B gene) in human macrophages decreased cellular lipid accumulation, the precipitating factor in foam cell formation. These results suggest that inhibition of 15-LOX-2 is a strategy for mitigating the development of cardiovascular disease.Lipoxygenases oxygenate arachidonic acid (AA) to form a stereo-and regiospecific isomer of hydroperoxyeicosatetraenoic acid (HpETE), and isoforms are named according to which carbon atom is oxygenated (for review see Refs. 7 and 8).In general, within a species, lipoxygenases that differ in regiospecificity share ϳ40% sequence identity. In contrast, betweenspecies LOX homologues are expected to share ϳ75% or better sequence identity and generate the same HpETE isomer. The mouse homologue of human 15-LOX-2, however, is an 8-LOX (m8S-LOX) with free AA as the substrate, and this altered regiospecificity appears to be unique to the mouse enzyme (9). We asked whether at the membrane and in a cellular context the mouse enzyme might also generate a 15-HpETE product. This information might prov...
Viral tropism within the brain and the role(s) of vertebrate immune response to neurotropic flaviviruses infection is largely understudied. We combine multimodal imaging (cm-nm scale) with single nuclei RNA-sequencing to study Langat virus in wildtype and interferon alpha/beta receptor knockout (Ifnar–/–) mice to visualize viral pathogenesis and define molecular mechanisms. Whole brain viral infection is imaged by Optical Projection Tomography coregistered to ex vivo MRI. Infection is limited to grey matter of sensory systems in wildtype mice, but extends into white matter, meninges and choroid plexus in Ifnar–/– mice. Cells in wildtype display strong type I and II IFN responses, likely due to Ifnb expressing astrocytes, infiltration of macrophages and Ifng-expressing CD8+ NK cells, whereas in Ifnar–/–, the absence of this response contributes to a shift in cellular tropism towards non-activated resident microglia. Multimodal imaging-transcriptomics exemplifies a powerful way to characterize mechanisms of viral pathogenesis and tropism.
Neurotropic virus infections cause tremendous disease burden. Methods visualizing infection in the whole brain remain unavailable which greatly impedes understanding of viral neurotropism and pathogenesis. We devised an approach to visualize the distribution of neurotropic virus infection in whole mouse brain ex vivo. Optical projection tomography (OPT) signal was coregistered with a unique magnetic resonance imaging (MRI) brain template, enabling precise anatomical mapping of viral distribution, and the effect of type I interferon on distribution of infection was analyzed. Guided by OPT-MR, we show that Langat virus specifically targets sensory brain systems and the lack of type I interferon response results in an anatomical shift in infection patterns in the brain. We confirm this regional tropism, observed with whole brain OPT-MRI, by confocal and electron microscopy to provide unprecedented insight into viral neurotropism. This approach can be applied to any fluorescently labeled target in the brain.
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