Biology subjects

Zarrow, J. E.

Publications and source records attributed to Zarrow, J. E..

3 recordsLinked to original sources

bioRxiv2024

N-Aldehyde-Modified Phosphatidylethanolamines generated by lipid peroxidation are robust substrates of N-Acyl Phosphatidylethanolamine Phospholipase D

N-acyl phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) hydrolyzes phosphatidylethanolamines (PE) where the headgroup nitrogen has been enzymatically modified with acyl chains of four carbons or longer (N-acyl-PEs or NAPEs). The nitrogen headgroup of PE can also be non-enzymatically modified by reactive lipid aldehydes, thus forming N-aldehyde modified-PEs (NALPEs). Some NALPEs such as N-carboxyacyl-PEs are linked to PE via amide bonds similar to NAPEs, but others are linked by imine, pyrrole, or lactam moieties. Whether NAPE-PLD can hydrolyze NALPEs was unknown. We therefore characterized the major NALPE species formed during lipid peroxidation of arachidonic acid and linoleic acid and generated various NALPEs for characterization of their sensitivity to NAPE-PLD hydrolysis by reacting synthesized aldehydes with PE. We found that NAPE-PLD could act on NALPEs of various lengths and linkage types including those derived from PE modified by malondialdehyde (N-MDA-PE), butane dialdehyde (N-BDA-PE), 4-hydroxynonenal (N-HNE-PE), 4-oxo-nonenal (N-ONE-PE), 9-keto-12-oxo-dodecenoic acid (N-KODA-PE), and 15-E2-isolevuglandin (N-IsoLG-PE). To assess the relative preference of NAPE-PLD for various NALPEs versus its canonical NAPE substrates, we generated a substrate mixture containing roughly equimolar concentrations of the seven NALPEs as well as two NAPEs (N-palmitoyl-PE and N-linoleoyl-PE) and measured their rate of hydrolysis. Several NALPE species, including the N-HNE-PE pyrrole species, were hydrolyzed at a similar rate as N-linoleoyl-PE and many of the other NALPEs showed intermediate rates of hydrolysis. These results significantly expand the substrate repertoire of NAPE-PLD and suggest that it may play an important role in clearing products of lipid peroxidation in addition to its established role in the biosynthesis of N-acyl-ethanolamines.

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↗