We previously demonstrated neutrophil MPO derived HOCl targets the vinyl ether bond of plasmalogens resulting in the Liberation of 2-chlorofatty aldehydes (2-ClFALDs) and their oxidation products, 2-chlorofatty acids (2-ClFAs), which elicit neutrophil extracellular trap (NET) formation. In this study, the click chemistry analog of 2-chlorohexadecanoic acid (2-ClHA) was utilized to identify 127 proteins covalently modified by 2-ClHA in human neutrophils. Bioinformatics revealed that multiple proteins modified by 2-ClHA are related to protein modification and binding as well as metabolite interconversion. Three key proteins involved in NET formation and function were modified by 2-ClHA including peptidyl arginine deiminase 4 (PAD4), neutrophil defensin alpha 3 (DEFA3), and neutrophil collagenase (MMP8). PAD4 activity was shown to be increased by 2-ClFA treatment. Further studies investigated 2-ClFA modified protein localization over time during NET formation. Initially PAD4 and 2-ClFA-modified proteins were extranuclear but over time they both localized to distinct nuclear regions. Following DNA release from neutrophils, 2-ClFA-modified proteins were found throughout the neutrophil and DNA strands. In summary, multiple neutrophil proteins are modified by 2-ClHA, including PAD4. 2-ClHA modification and activation of PAD4 is suggested as a key component of 2-ClHA elicited NET formation.

Genetic and dietary cues are known drivers of obesity, yet how they converge at the molecular level is incompletely understood. Here we show that PPARγ supports hypertrophic expansion of adipose tissue via transcriptional control of LPCAT3, an endoplasmic reticulum (ER)-resident O-acyltransferase that selectively enriches diet-derived omega-6 polyunsaturated fatty acids (n-6 PUFAs) in the membrane lipidome. In mice fed a high-fat diet, lowering membrane n-6 PUFA levels through genetic or dietary interventions results in aberrant adipose triglyceride (TG) turnover, ectopic fat deposition and insulin resistance. Additionally, we detail a non-canonical adaptive response in ‘lipodystrophic’ Lpcat3 adipose tissues that engages a futile lipid cycle to increase metabolic rate and offset lipid overflow to ectopic sites. Live-cell imaging, lipidomics and molecular dynamics simulations reveal that adipocyte LPCAT3 activity enriches n-6 arachidonate in the phosphatidylethanolamine (PE)-dense ER-lipid droplet interface. Functionally, this localized PE remodelling optimizes TG storage by driving the formation of large droplets that exhibit greater resistance to adipose TG lipase activity. These findings highlight the PPARγ-LPCAT3 axis as a mechanistic link between dietary n-6 PUFA intake, adipose expandability and systemic energy balance.

Several oxylipins are potent lipid mediators that regulate diverse aspects of health and disease and whose quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) presents substantial technical challenges. As members of the lipidomics community, we developed technical recommendations to ensure best practices when quantifying oxylipins by LC-MS.