Lipoprotein-associated phospholipase A2: A paradigm for allosteric regulation by membranes

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.

Oxidized Phospholipids Promote NETosis and Arterial Thrombosis in LNK(SH2B3) Deficiency

Background: LNK/SH2B3 inhibits JAK/STAT signaling by hematopoietic cytokine receptors. GWAS have shown association of a common SNP in LNK (R262W, T allele) with neutrophilia, thrombocytosis and coronary artery disease (CAD). We have shown that LNK(TT) reduces LNK function and that LNK deficient mice display prominent platelet−neutrophil aggregates, accelerated atherosclerosis and thrombosis. Platelet−neutrophil interactions can promote neutrophil extracellular trap (NET) formation. The goals of this study were to assess the role of neutrophil extracellular traps (NETs) in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency. Methods: We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme peptidylarginine deiminase4 (PAD4) and transplanted their bone marrow into Ldlr −/− mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2 V617F ). Results: Lnk deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated Lnk −/− platelets promoted increased NETosis when incubated with Lnk −/− neutrophils compared to WT platelets or WT neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from Lnk −/− platelets, as well as increased priming and response of Lnk −/− neutrophils to OxPL. To counteract the effects of OxPL, we introduced a transgene expressing the single-chain variable fragment of E06 (E06−scFv). E06−scFv reversed accelerated NETosis, atherosclerosis and thrombosis in Lnk −/− mice. We also showed increased NETosis when human induced pluripotent stem cell (iPSC) derived LNK(TT) neutrophils were incubated with LNK(TT) platelet/megakaryocytes, but not in isogenic LNK(CC) controls, confirming human relevance. Using data from UK Biobank we found that individuals with the JAK2 VF mutation only showed increased CAD when also carrying the LNK R262W allele. Mice with hematopoietic Lnk +/- and Jak2 VF clonal hematopoiesis, showed accelerated arterial thrombosis but not atherosclerosis compared to Jak2VFLnk +/+ controls. Conclusions: Hematopoietic Lnk deficiency promotes NETosis and arterial thrombosis in an OxPL-dependent fashion. LNK(R262W) reduces LNK function in human platelets and neutrophils promoting NETosis, and increases CAD risk in humans carrying JAK2 VF mutations. Therapies targeting OxPL may be beneficial for CAD in genetically defined human populations.

Oxidized Phospholipids in Control of Endothelial Barrier Function: Mechanisms and Implication in Lung Injury

Earlier studies investigating the pathogenesis of chronic vascular inflammation associated with atherosclerosis described pro-inflammatory and vascular barrier disruptive effects of lipid oxidation products accumulated in the sites of vascular lesion and atherosclerotic plaque. However, accumulating evidence including studies from our group suggests potent barrier protective and anti-inflammatory properties of certain oxidized phospholipids (OxPLs) in the lung vascular endothelium. Among these OxPLs, oxidized 1-palmitoyl-2-arachdonyl-sn-glycero-3-phosphocholine (OxPAPC) causes sustained enhancement of lung endothelial cell (EC) basal barrier properties and protects against vascular permeability induced by a wide variety of agonists ranging from bacterial pathogens and their cell wall components, endotoxins, thrombin, mechanical insults, and inflammatory cytokines. On the other hand, truncated OxPLs cause acute endothelial barrier disruption and potentiate inflammation. It appears that multiple signaling mechanisms triggering cytoskeletal remodeling are involved in OxPLs-mediated regulation of EC barrier. The promising vascular barrier protective and anti-inflammatory properties exhibited by OxPAPC and its particular components that have been established in the cellular and animal models of sepsis and acute lung injury has prompted consideration of OxPAPC as a prototype therapeutic molecule. In this review, we will summarize signaling and cytoskeletal mechanisms involved in OxPLs-mediated damage, rescue, and restoration of endothelial barrier in various pathophysiological settings and discuss a future potential of OxPAPC in treating lung disorders associated with endothelial barrier dysfunction.

A highly conserved host lipase deacylates oxidized phospholipids and ameliorates acute lung injury in mice

Oxidized phospholipids have diverse biological activities, many of which can be pathological, yet how they are inactivated in vivo is not fully understood. Here, we present evidence that a highly conserved host lipase, acyloxyacyl hydrolase (AOAH), can play a significant role in reducing the pro-inflammatory activities of two prominent products of phospholipid oxidation, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. AOAH removed the sn-2 and sn-1 acyl chains from both lipids and reduced their ability to induce macrophage inflammasome activation and cell death in vitro and acute lung injury in mice. In addition to transforming Gram-negative bacterial lipopolysaccharide from stimulus to inhibitor, its most studied activity, AOAH can inactivate these important danger-associated molecular pattern molecules and reduce tissue inflammation and injury.

What Does Time-Dependent Fluorescence Shift (TDFS) in Biomembranes (and Proteins) Report on?

The organization of biomolecules and bioassemblies is highly governed by the nature and extent of their interactions with water. These interactions are of high intricacy and a broad range of methods based on various principles have been introduced to characterize them. As these methods view the hydration phenomena differently (e.g., in terms of time and length scales), a detailed insight in each particular technique is to promote the overall understanding of the stunning “hydration world.” In this prospective mini-review we therefore critically examine time-dependent fluorescence shift (TDFS)—an experimental method with a high potential for studying the hydration in the biological systems. We demonstrate that TDFS is very useful especially for phospholipid bilayers for mapping the interfacial region formed by the hydrated lipid headgroups. TDFS, when properly applied, reports on the degree of hydration and mobility of the hydrated phospholipid segments in the close vicinity of the fluorophore embedded in the bilayer. Here, the interpretation of the recorded TDFS parameters are thoroughly discussed, also in the context of the findings obtained by other experimental techniques addressing the hydration phenomena (e.g., molecular dynamics simulations, NMR spectroscopy, scattering techniques, etc.). The differences in the interpretations of TDFS outputs between phospholipid biomembranes and proteins are also addressed. Additionally, prerequisites for the successful TDFS application are presented (i.e., the proper choice of fluorescence dye for TDFS studies, and TDFS instrumentation). Finally, the effects of ions and oxidized phospholipids on the bilayer organization and headgroup packing viewed from TDFS perspective are presented as application examples.

Oxidized Phospholipids and Neutrophil Elastase Coordinately Play Critical Roles in NET Formation

Neutrophil extracellular traps (NETs) are web-like structures consisting of decondensed chromatin DNA and contents of granules, such as myeloperoxidase (MPO) and neutrophil elastase (NE). NETs are usually released from neutrophils undergoing NETosis, a neutrophil-specific cell death mode characterized by the collapse and disappearance of cell membranes and nuclear envelopes. It is well known that production of reactive oxygen species (ROS) triggers NETosis and NET formation. However, details of intracellular signaling downstream of ROS production during NETosis and NET formation remains uncertain. Here, we demonstrated that the peroxidation of phospholipids plays a critical role in NETosis and NET formation induced by phorbol 12-myristate13-acetate (PMA) or immune complex in vitro and by lipopolysaccharide (LPS) in vivo. This phospholipid peroxidation is mediated by the enzymatic activity of MPO. On the other hand, NE, which was previously reported to be released from granules to cytosol by MPO during NET formation, is not required for either the peroxidation of phospholipids or the execution of NETosis, but contributes to chromatin decondensation and nuclear swelling independently of MPO-mediated oxidized phospholipids. Analysis of isolated nuclei clearly demonstrated that oxidized phospholipids and NE differently yet synergistically execute chromatin decondensation and nuclear swelling, and the subsequent release of nuclear contents. These findings indicate the dual roles of MPO in NETosis and NET formation, and provide new insight into the molecular mechanism of these phenomena.