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Inactivation of PAF-AH and nitration of tyrosine residues is prevented by PNU-101033E. A) Aliquots of Pafase (1.6 g containing 0.22 mol/min of PAF-AH activity) were treated with SIN-1 (0, 0.3, 1, 3.0, 10 mM) and either DMSO or PNU-101033E in DMSO (1-100 M) at pH 7.4 for 4 h at 37°C, in a total volume of 50 l. Ten-microliter aliquots were subjected to immunoblot analysis using an antinitrotyrosine antibody. B) Appropriate aliquots from (A) were assayed for PAF-AH activity.
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Platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase A2 that inactivates potent lipid messengers, such as PAF and modified phospholipids generated in settings of oxidant stress. The catalytic activity of PAF-AH is sensitive to oxidants, a feature that may have pathological consequences. We report that peroxynitrite, an oxidant spe...
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Context 1
... To investigate whether derivatization of one or more tyrosine residues by nitration-mediated inactiva- tion of PAF-AH, we tested the effect of PNU-101033E, a pyrrolopyrimidine antioxidant previously shown to spe- cifically block peroxynitrite-mediated tyrosine nitration (41). PNU-101033E (100 M) completely prevented tyrosine nitration of PAF-AH ( Fig. 2A), had no signifi- cant effect on enzymatic activity in the absence of SIN-1, and prevented oxidative inactivation at concentrations of SIN-1 up to 1 mM (Fig. 2B). These results indicated that a tyrosine residue(s) was a target of oxidative inactivation at relatively low oxidant levels and suggested that mecha- nisms other than tyrosine ...
Context 2
... antioxidant previously shown to spe- cifically block peroxynitrite-mediated tyrosine nitration (41). PNU-101033E (100 M) completely prevented tyrosine nitration of PAF-AH ( Fig. 2A), had no signifi- cant effect on enzymatic activity in the absence of SIN-1, and prevented oxidative inactivation at concentrations of SIN-1 up to 1 mM (Fig. 2B). These results indicated that a tyrosine residue(s) was a target of oxidative inactivation at relatively low oxidant levels and suggested that mecha- nisms other than tyrosine nitration contributed to enzy- matic inactivation at higher oxidant concentrations. This issue was addressed in experiments described in the next sections. A ...
Context 3
... of Y-84, -85, -103, -160, and -205 in the PSIV construct substantially increased susceptibility to oxidative inactivation (Supplemental Fig. 2S). The folding of these proteins may facilitate access of oxidants to target sites. Alternatively, these residues could have protective scav- enging functions in the wild-type ...
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... then oxidized the particles, separated the soluble and LDL fractions by ultracentrifugation, and subjected these extracts to enzymatic activity and immunoblot analyses using anti-PAF-AH antibodies. A control incubation conducted in the absence of oxidants showed that most of the PAF-AH protein remained associated with LDL throughout the procedure (Fig. 9, lanes 1 and 2 showing the soluble and LDL fractions, respectively). Treatment with SIN-1 attenuated association with LDL (Fig. 9, compare LDL fractions depicted in lanes 2, 4, and 6) and increased the amount of immunoreactive protein in the soluble fraction (Fig. 9, compare soluble fractions depicted in lanes 1, 3, and 5). These results indicated ...
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Citations
... Peroxynitrite, one of the main oxidants produced during in vivo cellular oxidation, was found to inhibit Lp-PLA2 (Gurung and Bhattacharjee, 2018b). A team of researchers discovered Met117, a critical target of protein oxidation, that was exposed on the protein surface using site-directed mutagenesis (MacRitchie et al., 2007). In addition to resulting in enzymatic inactivation, its oxidation would have a negative impact on the protein's capacity to interact with LDL. ...
Lipoprotein-associated phospholipase A2, a hydrophobic enzyme that is mainly secreted by macrophages, plays a role in the vascular inflammation that results in coronary artery disease and other vascular disorders. It has an antiinflammatory effect by hydrolyzing oxidized low-density lipoprotein and inactivating the proinflammatory platelet-activating factor. In addition, it has proinflammatory effects because it produces bioactive substances that promote inflammation, such as lysophosphatidylcholine and oxidized nonesterified fatty acids. Therefore, it is challenging to determine whether lipoprotein-associated phospholipase A2 promotes antiinflammatory pathways, promotes inflammation, or participates in both anti and proinflammatory processes in the pathogenesis of coronary artery disease. Additionally, clinicians are looking for novel biomarkers to distinguish between patients who are at low and high risk of developing coronary artery disease. Estimates of its plasma concentration may also be useful in comparing individuals with low and high risk of developing coronary artery disease. However, numerous clinical studies based on empirical evidence still need to be carried out in order to fully understand the pathophysiology of lipoprotein-associated phospholipase A2 at the onset of coronary artery disease.
... Both increased and decreased levels of PAF-AH are reported in diseases such as asthma, hypertension, systemic lupus erythematosus, atherosclerosis, sepsis, and Crohn's disease. One possible reason for this variation may be the susceptibility of PAF-AH to oxidative inactivation and the involvement of multiple mediators in regulating PAF-AH levels both at transcription and translation (59,60). Expression of the PAF-AH gene is negatively modulated by LPS and cytokines like INF-γ (61) and positively regulated by PAF, INF-α, IL-1α, IL-4, IL-6, TNF-α, and GM-CSF in monocyte-derived macrophages (61). ...
Platelet-activating factor (PAF), a bioactive ether phospholipid with significant pro-inflammatory properties, was identified almost half a century ago. Despite extensive study of this autocoid, therapeutic strategies for targeting its signaling components have not been successful, including the recent clinical trials with darapladib, a drug that targets plasma PAF-acetylhydrolase (PAF-AH). We recently provided experimental evidence that the previously unrecognized acyl analog of PAF, which is concomitantly produced along with PAF during bio-synthesis, dampens PAF signaling by acting both as a sacrificial substrate for PAF-AH and probably as an en-dogenous PAF-receptor antagonist/partial agonist. If this is the scenario in vivo, PAF-AH needs to catalyze the selective hydrolysis of alkyl-PAF and not acyl-PAF. Accordingly, different approaches are needed for treating inflammatory diseases in which PAF signaling is implicated. The interplay between acyl-PAF, alkyl-PAF, PAF-AH, and PAF-R is complex, and the outcome of this interplay has not been previously appreciated. In this review, we discuss this interaction based on our recent findings. It is very likely that the relative abundance of acyl and alkyl-PAF and their interactions with PAF-R in the presence of their hydrolyzing enzyme PAF-AH may exert a modulatory effect on PAF signaling during inflammation.
... Cells typically neutralize oxidative stress by upregulating the production of antioxidant enzymes such as catalase, superoxide dismutase, glutathione S-transferase, glutathione peroxidase, heme oxygenase-1, and NADPH-quinine oxidase [43]. Because PAF-AH is susceptible to oxidative inactivation [44], we believe that oxidative stress is a critical component in PAF accumulation and signaling. Previously, we showed that intraperitoneal injection of PAF caused sudden death in Swiss albino mice through the PAF-R [45,46]. ...
Platelet-activating factor (PAF) is a potent inflammatory agonist. In Swiss albino mice, intraperitoneal injection of PAF causes sudden death with oxidative stress and disseminated intravascular coagulation (DIC), characterized by prolonged prothrombin time, thrombocytopenia, reduced fibrinogen content, and increased levels of fibrinogen degradation products. However, the underlying mechanism(s) is unknown. The PAF-R antagonist WEB-2086 protected mice against PAF-induced death by reducing DIC and oxidative stress. Accordingly, general antioxidants such as ascorbic acid, α-tocopherol, gallic acid, and N-acetylcysteine partially protected mice from PAF-induced death. N-acetylcysteine, a clinically used antioxidant, prevented death in 67% of mice, ameliorated DIC characteristics and histological alterations in the liver, and reduced oxidative stress. WEB-2086 suppressed H2O2-mediated oxidative stress in isolated mouse peritoneal macrophages, suggesting that PAF signaling may be a downstream effector of reactive oxygen species generation. PAF stimulated all three (ERK, JNK, and p38) of the MAP-kinases, which were also inhibited by N-acetylcysteine. Furthermore, a JNK inhibitor (SP600125) and ERK inhibitor (SCH772984) partially protected mice against PAF-induced death, whereas a p38 MAP-kinase inhibitor (SB203580) provided complete protection against DIC and death. In human platelets, which have the canonical PAF-R and functional MAP-kinases, JNK and p38 inhibitors abolished PAF-induced platelet aggregation, but the ERK inhibitor was ineffective. Our studies identify p38 MAP-kinase as a critical, but unrecognized component in PAF-induced mortality in mice. These findings suggest an alternative therapeutic strategy to address PAF-mediated pathogenicity, which plays a role in a broad range of inflammatory diseases.
... Peroxynitrite, one of the key oxidants produced in cellular oxidation in vivo, was identified to inactivate Lp-PLA2. 45 Using site-directed mutagenesis, MacRitchie et al 46 revealed that a primary target of oxidation in the protein was Met117, which was exposed on the protein surface; ...
Inflammation is thought to play an important role in the pathogenesis of vascular diseases. Lipoprotein‐associated phospholipase A2 (Lp‐PLA2) mediates vascular inflammation through the regulation of lipid metabolism in blood, thus, it has been extensively investigated to identify its role in vascular inflammation‐related diseases, mainly atherosclerosis. Although darapladib, the most advanced Lp‐PLA2 inhibitor, failed to meet the primary endpoints of two large phase III trials in atherosclerosis patients cotreated with standard medical care, the research on Lp‐PLA2 has not been terminated. Novel pathogenic, epidemiologic, genetic, and crystallographic studies regarding Lp‐PLA2 have been reported recently, while novel inhibitors were identified through a fragment‐based lead discovery strategy. More strikingly, recent clinical and preclinical studies revealed that Lp‐PLA2 inhibition showed promising therapeutic effects in diabetic macular edema and Alzheimer's disease. In this review, we not only summarized the knowledge of Lp‐PLA2 established in the past decades but also emphasized new findings in recent years. We hope this review could be valuable for helping researchers acquire a much deeper insight into the nature of Lp‐PLA2, identify more potent and selective Lp‐PLA2 inhibitors, and discover the potential indications of Lp‐PLA2 inhibitors.
... In this context, Ambrosio et al. (1994) observed that reactive oxygen species (ROS)-induced inactivation of PAF-AH may be prevented completely by ROS scavenger and antioxidant enzymes. Similarly, MacRitchie et al. (2007) showed that peroxinitrite (ONOO -), a highly reactive species, causes enzyme inactivation due to tyrosine nitration. Both ONOO --and ROS-initiated stress can be attenuated by exenatide (Zhao et al., 2013) suggesting that exenatide may protect lipoprotein-associated PAF-AH against oxidative inactivation. ...
... Dynamic variation in this enzyme has been seen in both humans and experimental animal models (31). Moreover, the plasma form of PAF-AH is susceptible to oxidative modification (53), and it is regulated at the transcription level by multiple mediators (54), suggesting a dynamic role for this enzyme in maintaining levels of acyl-PAF, alkyl-PAF, and PAFmimetics. For this reason, we studied the effect of PAF-AH levels on dampening the activation of PAF-R in the presence of acyl-PAF by progressively increasing the circulating levels of PAF-AH to mimic the dynamic variation observed in both physiologic and pathologic settings (31). ...
Platelet-activating factor (alkyl-PAF) is a potent inflammatory mediator that exerts its actions via single PAF receptor (PAF-R). Cells that biosynthesize alkyl-PAF also make abundant amounts of the less potent PAF analog acyl-PAF, which competes for PAF-R. Both PAF species are degraded by plasma form of PAF acetylhydrolase (PAF-AH). We examined whether co-generated acyl-PAF protects alkyl-PAF from systemic degradation by acting as a sacrificial substrate to enhance inflammatory stimulation or if it acts as an inhibitor to dampen PAF-R signaling. Both PAF species are prothrombotic in isolation, but acyl-PAF reduced the alkyl-PAF stimulation of human platelets expressing canonical PAF-R. In Swiss albino mice, alkyl-PAF causes sudden death, but this effect can also be suppressed by simultaneously administering boluses of acyl-PAF. When PAF-AH levels were incrementally elevated, the protective effect of acyl-PAF on alkyl-PAF-induced death was serially decreased. We conclude that although acyl-PAF in isolation is mildly proinflammatory, in a pathophysiological setting, abundant acyl-PAF suppresses functions of alkyl-PAF. These studies provide, an unrecognized role for acyl-PAF, which acts as an inflammatory set-point modulator that regulates both PAF-R signaling and hydrolysis.
... One of the key oxidant produced in cellular oxidation in vivo is peroxynitrite that inactivates Lp-PLA 2 . Earlier report suggests that the surface exposed Met117 is a primary target of oxidation in the protein [15]. Fig. 1A shows the position of Met117 in Lp-PLA 2 structure and Fig. 1B represents the chemical structure of Methionine sulfoxide (MetSO) generated upon oxidation of Methionine residue. ...
... Fig. 1A shows the position of Met117 in Lp-PLA 2 structure and Fig. 1B represents the chemical structure of Methionine sulfoxide (MetSO) generated upon oxidation of Methionine residue. Met117 oxidation besides enzymatic inactivation also affects its association with LDL perhaps due to the proximity of this residue to the LDL binding domain [13,15]. ...
... The RMSF values of Met117 increased from 0.2036 nm to 0.2155 nm upon oxidation. It was reported earlier that oxidation of Met117 impairs the ability of Lp-PLA 2 to associate with LDL particles because of proximity of Met117 to key LDL binding residues such as Trp115 and Leu116 [15]. Thus, the increased structural flexibility of the LDL binding region upon Met117 oxidation explains its inability to bind with LDL particles. ...
Human Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an important biomarker for cardiovascular diseases and a therapeutically important drug target against Atherosclerosis. It has the ability to hydrolyze various oxidized low density lipoproteins (LDL) and generates potent pro-inflammatory signaling molecules. Both physiological and non-physiological oxidants have been reported to inhibit Lp-PLA2 activity. The mechanism of the enzyme inhibition due to oxidation of surface exposed Met117 at the structural level is not clearly understood. In the present work, molecular dynamics (MD) simulation and Essential dynamics (ED) has been used in tandem with molecular docking approach to understand the structural alteration in Lp-PLA2 upon Met117 oxidation. Further, the binding of substrate, Platelet-activating factor (PAF) with the wild type and oxidized form have also been investigated. Our results showed that Met117 oxidation caused enhanced flexibility and decreased compactness in oxidized state. PAF binding interaction with oxidized protein was mediated only through hydrophobic interactions. MD simulation studies revealed that the oxidized protein failed to firmly bind PAF. Our present findings will help understand the mechanism of Lp-PLA2 inhibition under oxidative stress.
... It was reported earlier that replacement of Tyr307 and Tyr335 by phenylalanine residues in Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) results in increased resistance to high oxidant levels. This lends support for these residues as easy targets for nitration in physiological settings which lead to oxidative inactivation of Lp-PLA 2 enzyme [19]. Lp-PLA 2 belongs to secretory Phospholipase A 2 group VII and was discovered because of its ability to hydrolyze and inactivate a potent pro-inflammatory signaling molecule, Platelet Activating Factor (PAF). ...
Protein tyrosine nitration (PTN) is a post translational event which results in the generation of 3-Nitrotyrosine (3-NT). High levels of 3-NT were reported in several human diseases such as Parkinson's disease, Alzheimer's disease, amylotrophic lateral sclerosis and coronary artery disease. It was reported that PTN at positions 307 and 335 of Lipoprotein-associated phospholipase A2 (Lp-PLA2) curtails its enzymatic activity but the mechanism of inhibition at the structure level is still incomprehensible. The present study is an in silico endeavor to understand nitrative stress induced structural changes in Lp-PLA2. Molecular docking studies revealed a decreased binding affinity of substrate, Platelet Activating Factor (PAF) with the nitrated forms of Lp-PLA2 (NT-Tyr307 and NT-Tyr335) compared to the wild type, due to differences in the hydrogen bond interaction patterns. Molecular dynamics (MD) simulation studies suggests higher flexibility of nitrated forms compared to wild type, disorientation of the catalytic triad and decreased molecular interactions of NT-Tyr307 and NT-Tyr335 with other residues of the protein. Essential dynamics (ED) further confirmed the enhanced structural flexibility of nitrated forms of Lp-PLA2. Our findings would help understand the molecular mechanism of nitrative stress induced inhibition of Lp-PLA2 which may further assist in designing of therapeutics having protective functions against PTN.
... Due to the property of Lp-PLA2, it accumulates lysophosphatidylcholine in situ and induces the expression of several inflammatory cytokines [13]. MacRitchie [14] reports that Lp-PLA2 inactivates potent lipid messengers, such as PAF and modified phospholipids in settings of oxidant stress, and has the susceptibility to oxidative attack. In hypercholesterolemic pigs with high fat diet, De Keyzer points out that increased levels of oxidized LDL coming along with obvious increase in Lp-PLA2 activity [15]. ...
... The plasma PAF-AH is susceptible to oxidant attack and suffers inactivation from modification of the residues that contribute to enzymatic activity (75). Whether or not highly variable levels of circulating PAF-AH arise from oxidation in the general population, is not currently known. ...
Mounting ambiguity persists around the functional role of the plasma form of platelet-activating factor acetylhydrolase (PAF-AH). Since PAF-AH hydrolyses PAF and related oxidized phospholipids, it is widely accepted as an anti-inflammatory enzyme. On the other hand, its actions can also generate lysophosphatidylcholine (lysoPC) - a component of bioactive atherogenic oxidized LDL, thus allowing the enzyme to have pro-inflammatory capabilities. Presence of a canonical lysoPC receptor has been seriously questioned for a multitude of reasons. Animal models of inflammation show that elevating PAF-AH levels is beneficial and not deleterious and overexpression of PAF-R also augments inflammatory responses. Further, many Asian populations have a catalytically inert PAF-AH that appears to be a severity factor in a range of inflammatory disorders. Correlation found with elevated levels of PAF-AH and CVDs has led to the design of a specific PAF-AH inhibitor -Darapladib. However, in a recently concluded phase III STABILITY clinical trial, use of darapladib did not yield promising results. Presence of structurally related multiple ligands for PAF-R with varied potency, existence of multi-molecular forms of PAF-AH, broad substrate specificity of the enzyme and continuous PAF-production by the so called Bi-cycle of PAF makes PAF more enigmatic. This review seeks to address the above concerns.
