Lipoprotein-associated phospholipase A2 (Lp-PLA2) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein’s phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA2 binding to a phospholipid surface. This allosteric regulation of an enzyme’s activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA2 and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the sn-2 position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA2‘s specificity toward oxidized phospholipids.
Significant efforts have been made to ameliorate diabetic nephropathy (DN) by inhibiting protein kinase C. However, these efforts have not been successful in human trials, suggesting that novel therapeutic strategies are required. Thus far, it has been reported that green tea polyphenol epigallocatechin gallate (EGCg) improved albuminuria in DN in a human trial. Our previous study revealed that activation of diacylglycerol kinase α (DGKα) plays a crucial role in the amelioration of DN and that EGCg activates DGKα. Here, we investigated whether and how DGKα contributes to the amelioration of DN upon stimulation by EGCg by using streptozotocin-induced type 1 diabetic model mice. Our results revealed that EGCg ameliorated albuminuria in DN through DGKα in vivo, and methylated EGCg, which has higher absorption in the plasma improved albuminuria in DN effectively. Additionally, we showed that c-Src mediated EGCg-induced DGKα translocation and colocalized with the 67 kDa laminin receptor, which is an EGCg receptor. Furthermore, EGCg attenuated the loss of podocytes in DN by preventing a decrease in focal adhesion under high glucose conditions. Our results indicate that the DGKα pathway is an attractive therapeutic target and that activating this pathway is a novel strategy for treating DN.
Diabetic nephropathy (DN) is one of vascular complications of diabetes and is caused by abnormal protein kinase C activation as a result of increased diacylglycerol (DG) production in diabetic hyperglycaemia. Diacylglycerol kinase (DGK) converts DG into phosphatidic acid. Therefore, it is expected that the activation of DGK would ameliorate DN. Indeed, it has been reported that vitamin E (VtE) ameliorates DN in rat by activating DGK, and we recently reported that VtE specifically activates DGKα isoform in vitro. However, whether DGKα is involved in the VtE-induced amelioration of DN in vivo remains unknown. Therefore, we investigated the VtE-induced amelioration of DN in wild-type (DGKα+/+) and DGKα–deficient (DGKα−/−) mice in which diabetes was induced by streptozocin. Several symptoms of DN were ameliorated by VtE treatment in the DGKα+/+ mice but not in the DGKα−/− mice. Moreover, transmission electron microscopy of glomeruli and immunofluorescent staining of glomerular epithelial cells (podocytes) indicated that VtE ameliorates podocyte pathology and prevents podocyte loss in the DGKα+/+ mice but not in the DGKα−/− mice. We showed that VtE can ameliorate DN in mice and that DGKα is involved in the VtE-induced amelioration of DN in vivo, suggesting that DGKα is an attractive therapeutic target for DN.
Human phospholipase A 2 s (PLA 2 ) constitute a superfamily of enzymes that hydrolyze the sn -2 acyl-chain of glycerophospholipids, producing lysophospholipids and free fatty acids. Each PLA 2 enzyme type contributes to specific biological functions based on its expression, subcellular localization, and substrate specificity. Among the PLA 2 superfamily, the cytosolic cPLA 2 enzymes, calcium-independent iPLA 2 enzymes, and secreted sPLA 2 enzymes are implicated in many diseases, but a central issue is the preference for double-bond positions in polyunsaturated fatty acids (PUFAs) occupying the sn -2 position of membrane phospholipids. We demonstrate that each PLA 2 has a unique preference between the specific omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the omega-6 arachidonic acid (AA), which are the precursors of most proinflammatory and anti-inflammatory or resolving eicosanoids and related oxylipins. Surprisingly, we discovered that human cPLA 2 selectively prefers AA, whereas iPLA 2 prefers EPA, and sPLA 2 prefers DHA as substrate. We determined the optimal binding of each phospholipid substrate in the active site of each PLA 2 to explain these specificities. To investigate this, we utilized recently developed lipidomics-based LC-MS/MS and GC/MS assays to determine the sn -2 acyl chain specificity in mixtures of phospholipids. We performed μs timescale molecular dynamics (MD) simulations to reveal unique active site properties, especially how the precise hydrophobic cavity accommodation of the sn -2 acyl chain contributes to the stability of substrate binding and the specificity of each PLA 2 for AA, EPA, or DHA. This study provides the first comprehensive picture of the unique substrate selectivity of each PLA 2 for omega-3 and omega-6 fatty acids.
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