Biology subjects

Mashhadi, Z.

Publications and source records attributed to Mashhadi, Z..

4 recordsLinked to original sources

bioRxiv2026

N-aldehyde modified phosphatidylethanolamines formed by lipid peroxidation inhibit efferocytosis.

Objective: Impairments in efferocytosis, the phagocytic clearance of apoptotic cells, contribute to the development of chronic inflammation and promote the formation of large necrotic cores within atherosclerotic plaques. A better understanding of the factors that impair efferocytosis could lead to improved interventions. Many chronic inflammatory conditions feature excessive lipid peroxidation, which generates reactive lipid aldehydes that can potentially form N-aldehyde-modified phosphatidylethanolamines (NAMPs). We therefore investigated the extent to which levels of NAMPs are elevated under conditions of oxidative stress and whether exposure of macrophages to NAMPs inhibit efferocytosis. Approach and Results: The effects of synthetic NAMPs and lipid peroxidation on efferocytosis were measured using the Incucyte Live-Cell Analysis System to monitor rate of ingestion of CypHer-labeled apoptotic cells by cultured bone marrow-derived macrophages in real time. The extent of NAMP formation under various conditions was examined using liquid chromatography coupled with mass spectrometry. Several representative species of NAMPs (N-isolevuglandin-phosphatidylethanolamine, N-IsoLG-PE; N-4-hydroxynonenal-phosphatidylethanolamine, N-HNE-PE; and N-azeloyl-phosphatidylethanolamine, N-Aze-PE) inhibited efferocytosis in a concentration-dependent manner. Hydrolysis of NAMPs by recombinant N-acyl phosphatidylethanolamine hydrolyzing phospholipase D (NAPE-PLD) abolished their inhibitory activity, whereas genetic deletion of macrophage NAPE-PLD enhanced NAMP-mediated inhibition of efferocytosis. Blocking the inhibitor of lipid peroxidation, glutathione peroxidase 4 (GPX4), increased macrophage NAMP concentrations and impaired efferocytosis. Unmodified high-density lipoprotein (HDL) promoted efferocytosis, but exposing HDL to reactive lipid aldehydes or peroxidizing agents markedly increased NAMPs and this modified HDL inhibited efferocytosis. HDL isolated from subjects with familial hypercholesterolemia had elevated levels of multiple NAMP species. NAMPs impaired macrophage cholesterol efflux, which is required for continual efferocytosis. Conclusion: NAMPs accumulate under conditions associated with lipid peroxidation and inhibit the ability of macrophages to carry out efferocytosis.

immunology↗

bioRxiv2024

Plaat1l1 controls feeding induced NAPE biosynthesis and contributes to energy balance regulation in zebrafish.

Dysregulation of energy balance leading to obesity is a significant risk factor for cardiometabolic diseases such as diabetes, non-alcoholic fatty liver disease and atherosclerosis. In rodents and a number of other vertebrates, feeding has been shown to induce a rapid rise in the intestinal levels of N-acyl-ethanolamines (NAEs) and the chronic consumption of a high fat diet abolishes this rise. Administering NAEs to rodents consuming a high fat diet reduces their adiposity, in part by reducing food intake and enhancing fat oxidation, so that feeding-induced intestinal NAE biosynthesis appears to be critical to appropriate regulation of energy balance. However, the contribution of feeding-induced intestinal NAE biosynthesis to appropriate energy balance remains poorly understood because the specific enzymes responsible for feeding-induced NAE biosynthesis have not been identified. The rate-limiting step in the intestinal biosynthesis of NAEs is formation of their immediate precursors, the N-acyl-phosphatidylethanolamines (NAPEs), by phosphatidylethanolamine N-acyltransferases (NATs). At least six NATs are found in humans and multiple homologs of these NATs are found in most vertebrate species. In recent years, the fecundity and small size of zebrafish (Danio rerio), as well as their similarities in feeding behavior and energy balance regulation with mammals, have led to their use to model key features of cardiometabolic disease. We therefore searched the Danio rerio genome to identify all NAT homologs and found two additional NAT homologs besides the previously reported plaat1, rarres3, and rarres3l, and used CRISPR/cas9 to delete these two NAT homologs (plaat1l1 and plaat1l2). While wild-type fish markedly increased their intestinal NAPE and NAE levels in response to a meal after fasting, this response was completely ablated in plaat1l1-/- fish. Furthermore, plaat1l1-/-fish fed a standard flake diet had increased weight gain and glucose intolerance compared to wild-type fish. The results support a critical role for feeding-induced NAE biosynthesis in regulating energy balance and suggest that restoring this response in obese animals could potentially be used to treat obesity and cardiometabolic disease.

biochemistry↗

bioRxiv2023

Small Molecule Activation of NAPE-PLD Enhances Efferocytosis by Macrophages

N-acyl-phosphatidylethanolamine hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase that hydrolyzes N-acyl-phosphatidylethanolamine (NAPEs) to form N-acyl-ethanolamides (NAEs) and phosphatidic acid. Several lines of evidence suggest that reduced NAPE-PLD activity could contribute to cardiometabolic diseases. For instance, NAPEPLD expression is reduced in human coronary arteries with unstable atherosclerotic lesions, defective efferocytosis is implicated in the enlargement of necrotic cores of these lesions, and NAPE-PLD products such as palmitoylethanolamide and oleoylethanolamide have been shown to enhance efferocytosis. Thus, enzyme activation mediated by a small molecule may serve as a therapeutic treatment for cardiometabolic diseases. As a proof-of-concept study, we sought to identify small molecule activators of NAPE-PLD. High-throughput screening followed by hit validation and primary lead optimization studies identified a series of benzothiazole phenylsulfonyl-piperidine carboxamides that variably increased activity of both mouse and human NAPE-PLD. From this set of small molecules, two NAPE-PLD activators (VU534 and VU533) were shown to increase efferocytosis by bone-marrow derived macrophages isolated from wild-type mice, while efferocytosis was significantly reduced in Napepld-/- BMDM or after Nape-pld inhibition. Together these studies demonstrate an essential role for NAPE-PLD in the regulation of efferocytosis and the potential value of NAPE-PLD activators as a strategy to treat cardiometabolic diseases.

biochemistry↗

bioRxiv2020

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D

N-acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides (NAEs). Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a major obstacle to elucidating the role of NAPE-PLD and NAE biosynthesis in various physiological processes has been the lack of effective NAPE-PLD inhibitors. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (IC50 68 M) but LCA is also a highly potent ligand for TGR5 (EC50 0.52 M). Recently, the first selective small molecule inhibitor of NAPE-PLD, ARN19874, was reported (IC50 34 M). To identify more potent inhibitors of NAPE-PLD, we screened compounds using a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse NAPE-PLD. Screened compounds included a panel of bile acids as well as a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited NAPE-PLD with potency similar to LCA. Fourteen potent NAPE-PLD inhibitors were identified in the Spectrum Collection, with the two most potent (IC50 ~2 M) being symmetrically substituted dichlorophenes: hexachlorophene and bithionol. Structure activity relationship assays using additional substituted dichlorophenes identified key moieties needed for NAPE-PLD inhibition. Both hexachlorophene and bithionol showed significant selectivity for NAPE-PLD compared to non-target lipase activities such as S. chromofuscus PLD activity or serum lipase activity. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells.

biochemistry↗