PEX3 promotes regenerative repair after myocardial injury in mice through facilitating plasma membrane localization of ITGB3.

The peroxisome is a versatile organelle that performs diverse metabolic functions. PEX3, a critical regulator of the peroxisome, participates in various biological processes associated with the peroxisome. Whether PEX3 is involved in peroxisome-related redox homeostasis and myocardial regenerative repair remains elusive. We investigate that cardiomyocyte-specific PEX3 knockout (Pex3-KO) results in an imbalance of redox homeostasis and disrupts the endogenous proliferation/development at different times and spatial locations. Using Pex3-KO mice and myocardium-targeted intervention approaches, the effects of PEX3 on myocardial regenerative repair during both physiological and pathological stages are explored. Mechanistically, lipid metabolomics reveals that PEX3 promotes myocardial regenerative repair by affecting plasmalogen metabolism. Further, we find that PEX3-regulated plasmalogen activates the AKT/GSK3ß signaling pathway via the plasma membrane localization of ITGB3. Our study indicates that PEX3 may represent a novel therapeutic target for myocardial regenerative repair following injury.

High coverage of targeted lipidomics revealed lipid changes in the follicular fluid of patients with insulin-resistant polycystic ovary syndrome and a positive correlation between plasmalogens and oocyte quality.

Background: Polycystic ovary syndrome with insulin resistance (PCOS-IR) is the most common endocrine and metabolic disease in women of reproductive age, and low fertility in PCOS patients may be associated with oocyte quality; however, the molecular mechanism through which PCOS-IR affects oocyte quality remains unknown. Methods: A total of 22 women with PCOS-IR and 23 women without polycystic ovary syndrome (control) who underwent in vitro fertilization and embryo transfer were recruited, and clinical information pertaining to oocyte quality was analyzed. Lipid components of follicular fluid (FF) were detected using high-coverage targeted lipidomics, which identified 344 lipid species belonging to 19 lipid classes. The exact lipid species associated with oocyte quality were identified. Results: The number (rate) of two pronuclear (2PN) zygotes, the number (rate) of 2PN cleaved embryos, and the number of high-quality embryos were significantly lower in the PCOS-IR group. A total of 19 individual lipid classes and 344 lipid species were identified and quantified. The concentrations of the 19 lipid species in the normal follicular fluid (control) ranged between 10-3 mol/L and 10-9 mol/L. In addition, 39 lipid species were significantly reduced in the PCOS-IR group, among which plasmalogens were positively correlated with oocyte quality. Conclusions: This study measured the levels of various lipids in follicular fluid, identified a significantly altered lipid profile in the FF of PCOS-IR patients, and established a correlation between poor oocyte quality and plasmalogens in PCOS-IR patients. These findings have contributed to the development of plasmalogen replacement therapy to enhance oocyte quality and have improved culture medium formulations for oocyte in vitro maturation (IVM).

Distinctive Lipid Characteristics of Colorectal Cancer Revealed through Non-targeted Lipidomics Analysis of Tongue Coating.

The metabolites and microbiota in tongue coating display distinct characteristics in certain digestive disorders, yet their relationship with colorectal cancer (CRC) remains unexplored. Here, we employed liquid chromatography coupled with tandem mass spectrometry to analyze the lipid composition of tongue coating using a nontargeted approach in 30 individuals with colorectal adenomas (CRA), 32 with CRC, and 30 healthy controls (HC). We identified 21 tongue coating lipids that effectively distinguished CRC from HC (AUC = 0.89), and 9 lipids that differentiated CRC from CRA (AUC = 0.9). Furthermore, we observed significant alterations in the tongue coating lipid composition in the CRC group compared to HC/CRA groups. As the adenoma-cancer sequence progressed, there was an increase in long-chain unsaturated triglycerides (TG) levels and a decrease in phosphatidylethanolamine plasmalogen (PE-P) levels. Furthermore, we noted a positive correlation between N-acyl ornithine (NAOrn), sphingomyelin (SM), and ceramide phosphoethanolamine (PE-Cer), potentially produced by members of the Bacteroidetes phylum. The levels of inflammatory lipid metabolite 12-HETE showed a decreasing trend with colorectal tumor progression, indicating the potential involvement of tongue coating microbiota and tumor immune regulation in early CRC development. Our findings highlight the potential utility of tongue coating lipid analysis as a noninvasive tool for CRC diagnosis.

Temperature effects on plasmalogen profile and quality characteristics in Pacific oyster (Crassostrea gigas) during depuration.

The quality of Pacific oyster (Crassostrea gigas) can be affected by many factors during depuration, in which temperature is the major element. In this study, we aim to determine the quality and plasmalogen changes in C. gigas depurated at different temperatures. The quality was significantly affected by temperature, represented by varying survival rate, glycogen content, total antioxidant capacity, alkaline phosphatase activity between control and stressed groups. Targeted MS analysis demonstrated that plasmalogen profile was significantly changed during depuration with PUFA-containing plasmalogen species being most affected by temperature. Proteomics analysis and gene expression assay further verified that plasmalogen metabolism is regulated by temperature, specifically, the plasmalogen synthesis enzyme EPT1 was significantly downregulated by high temperature and four plasmalogen-related genes (GPDH, PEDS, Pex11, and PLD1) were transcriptionally regulated. The positive correlations between the plasmalogen level and quality characteristics suggested plasmalogen could be regarded as a quality indicator of oysters during depuration.

Selenoprotein I is indispensable for ether lipid homeostasis and proper myelination.

Selenoprotein I (SELENOI) catalyzes the final reaction of the CDP-ethanolamine branch of the Kennedy pathway, generating the phospholipids phosphatidylethanolamine (PE) and plasmenyl-PE. Plasmenyl-PE is a key component of myelin and is characterized by a vinyl ether bond that preferentially reacts with oxidants, thus serves as a sacrificial antioxidant. In humans, multiple loss-of-function mutations in genes affecting plasmenyl-PE metabolism have been implicated in hereditary spastic paraplegia, including SELENOI. Herein, we developed a mouse model of nervous system-restricted SELENOI deficiency that circumvents embryonic lethality caused by constitutive deletion and recapitulates phenotypic features of hereditary spastic paraplegia. Resulting mice exhibited pronounced alterations in brain lipid composition, which coincided with motor deficits and neuropathology including hypomyelination, elevated reactive gliosis, and microcephaly. Further studies revealed increased lipid peroxidation in oligodendrocyte lineage cells and disrupted oligodendrocyte maturation both in vivo and in vitro. Altogether, these findings detail a critical role for SELENOI-derived plasmenyl-PE in myelination that is of paramount importance for neurodevelopment.

Sea cucumber plasmalogen enhance lipophagy to alleviate abnormal lipid accumulation induced by high-fat diet.

Sea cucumber phospholipids, including the plasmalogen (PlsEtn) and plasmanylcholine (PakCho), have been shown to play a regulatory role in lipid metabolism disorders, but their mechanism of action remains unclear. Therefore, high-fat diet (HFD) and palmitic acid were used to establish lipid accumulation models in mice and HepG2 cells, respectively. Results showed that PlsEtn can reduce lipid deposition both in vivo and in vitro. HFD stimulation abnormally activated lipophagy through the phosphorylation of the AMPK/ULK1 pathway. The lipophagy flux monitor revealed abnormalities in the fusion stage of lipophagy. Of note, only PlsEtn stimulated the dynamic remodeling of the autophagosome membrane, which was indicated by the significantly decreased LC3 II/I ratio and p62 level. In all experiments, the effect of PlsEtn was significantly higher than that of PakCho. These findings elucidated the mechanism of PlsEtn in alleviating lipid accumulation, showed that it might be a lipophagy enhancer, and provided new insights into the high-value utilization of sea cucumber as an agricultural resource.

Plasmalogens Alter the Aggregation Rate of Transthyretin and Lower Toxicity of Transthyretin Fibrils.

Heart tissue can experience a progressive accumulation of transthyretin (TTR), a small four subunit protein that transports holoretinol binding protein and thyroxine. This severe pathology is known as transthyretin amyloid cardiomyopathy. Numerous experimental studies indicated that the aggregation rate and toxicity of TTR fibrils could be altered by the presence of lipids; however, the role of plasmalogens in this process remains unknown. In this study, we investigate the effect of choline plasmalogens (CPs) with different lengths and saturations of fatty acids (FAs) on TTR aggregation. We found that CPs with saturated and unsaturated FAs strongly suppressed TTR aggregation. We also found that CPs with saturated FAs did not change the morphology of TTR fibrils; however, much thicker fibrillar species were formed in the presence of CPs with unsaturated FAs. Finally, we found that CPs with C16:0, C18:0, and C18:1 FAs substantially lowered the cytotoxicity of TTR fibrils that were formed in their presence.

Sea Cucumber Plasmalogen Regulates the Lipid Profile in High-Fat Diet Mouse Liver via Lipophagy.

The sea cucumber plasmalogen PlsEtn has been shown to be associated with various chronic diseases related to lipid metabolism. However, the mechanism is unclear. Therefore, the present study used the sea cucumber plasmanylcholine PakCho as a structural contrast to PlsEtn and assessed its effect in 8 week high-fat diet (HFD)-fed mice. The lipidomic approach based on high-resolution mass spectrometry combined with molecular biology techniques was used to evaluate the mechanism of PlsEtn. The results showed that both PlsEtn and PakCho significantly inhibited an increase in mouse body weight and liver total triglyceride and total cholesterol levels caused by HFD. In addition, oil red O staining demonstrated that lipid droplets stored in the liver were degraded. Meanwhile, untargeted lipidomic experiments revealed that total lipids (increased by 42.8 mmol/mg prot; p < 0.05), triglycerides (increased by 38.9 mmol/mg prot; p < 0.01), sphingolipids (increased by 1.5 mmol/mg prot; p < 0.0001), and phospholipids (increased by 2.5 mmol/mg prot; p < 0.05) were all significantly elevated under HFD. PlsEtn resolved lipid metabolism disorders by alleviating the abnormal expression of lipid subclasses. In addition, five lipid molecular species, PE (18:1/20:4), PE (18:1/20:3), PE (18:1/18:3), TG (16:0/16:0/17:0), and TG (15:0/16:0/18:1), were identified as the biomarkers of HFD-induced lipid metabolism disorders. Finally, lipophagy-associated protein expression analysis showed that HFD abnormally activated lipophagy via ULK1 phosphorylation and PlsEtn alleviated lipophagy disorder through lysosomal function promotion. In addition, PlsEtn performed better than PakCho. Taken together, the current study results unraveled the mechanism of PlsEtn in alleviating lipid metabolism disorder and offered a new theoretical foundation for the high-value development of sea cucumber.

Plasmalogen, a glycerophospholipid crucial for Streptococcus mutans acid tolerance and colonization.

Plasmalogen is a specific glycerophospholipid present in both animal and bacterial organisms. It plays a crucial function in eukaryotic cellular processes and is closely related to several human diseases, including neurological disorders and cancers. Nonetheless, the precise biological role of plasmalogen in bacteria is not well understood. In this study, we identified SMU_438c as the enzyme responsible for plasmalogen production in Streptococcus mutans under anaerobic conditions. The heterologous expression of SMU_438c in a plasmalogen-negative strain, Streptococcus sanguinis, resulted in the production of plasmalogen, indicating that this enzyme is sufficient for plasmalogen production. Additionally, the plasmalogen-deficient S. mutans exhibited significantly lower acid tolerance and diminished its colonization in Drosophila flies compared to the wild-type strain and complemented strain. In summary, our data suggest that plasmalogen plays a vital role in bacterial stress tolerance and in vivo colonization. IMPORTANCE: This study sheds light on the biological role of plasmalogen, a specific glycerophospholipid, in bacteria, particularly in Streptococcus mutans. Plasmalogens are known for their significant roles in eukaryotic cells and have been linked to human diseases like neurological disorders and cancers. The enzyme SMU_438c, identified as essential for plasmalogen production under anaerobic conditions, was crucial for acid tolerance and in vivo colonization in Drosophila by S. mutans, underscoring its importance in bacterial stress response and colonization. These findings bridge the knowledge gap in bacterial physiology, highlighting plasmalogen's role in microbial survival and offering potential insights into microbial pathogenesis and host-microbe interactions.

Neuroprotective effect of plasmalogens on AlCl3-induced Alzheimer's disease zebrafish via acting on the regulatory network of ferroptosis, apoptosis and synaptic neurotransmission release with oxidative stress as the center.

Plasmalogens (Pls) are considered to play a potential role in the treatment of neurodegenerative diseases. In the present study, an Alzheimer's disease (AD) model of zebrafish induced by AlCl3 was established to investigate whether the marine-derived Pls could alleviate cognitive impairments of AD zebrafish. Behavioral tests were carried out to assess the athletic ability. The transcriptional profiles of zebrafish in the control, AD model and AD_PLS group were compared and analyzed to determine the potential mechanisms of dietary Pls on AD. The study found that Pls could reverse athletic impairment in the AD zebrafish model, and the expression levels of genes related to ferroptosis, synaptic dysfunction and apoptosis were significantly altered between experimental groups. Further analysis showed that all of these genes were associated with oxidative stress (OS). These data suggest that healthy protective role of marine-derived Pls on AD zebrafish may result from inhibition of ferroptosis and neuronal apoptosis, restoring synaptic neurotransmission release, and reducing neuroinflammation. Among them, Oxidative stress is acted as the center to connect different regulation pathways. This study provides evidence to support the essential roles of OS in pathogenesis of AD, and the application of Pls in relieving AD.

CSE triggers ferroptosis via SIRT4-mediated GNPAT deacetylation in the pathogenesis of COPD.

BACKGROUND: It is now understood that ferroptosis plays a significant role in the progression of chronic obstructive pulmonary disease (COPD) induced by cigarette smoke extract (CSE). However, the mechanisms underlying this relationship remain largely unclear. METHODS: In this study, we established a COPD mouse model through exposure to cigarette smoke particulates, followed by H&E staining, analysis of bronchoalveolar lavage fluid, and immunohistochemistry assay. A549 cells were exposed to increasing concentrations of CSE, with the addition of the ferroptosis activator erastin or the inhibitor Fer-1. Cell viability, LDH (lactate dehydrogenase) release, inflammatory cytokines, total ROS (reactive oxygen species), and lipid ROS were measured using the corresponding assay kits. The acetylation level of GNPAT was determined through immunoprecipitation. We assessed the expression levels of molecules involved in plasmalogen biosynthesis (FAR1, AGPS, and GNPAT), GPX4, and SIRT4 using quantitative real-time PCR, western blot analysis, and immunofluorescence staining. RESULTS: CSE-induced lung tissue damage was initially observed, accompanied by oxidative stress, ferroptosis, and increased plasmalogen biosynthesis molecules (FAR1, AGPS, and GNPAT). CSE also induced ferroptosis in A549 cells, resulting in reduced cell viability, GSH, and GPX4 levels, along with increased LDH, ROS, MDA (malondialdehyde) levels, oxidized lipids, and elevated FAR1, AGPS, and GNPAT expression. Knockdown of GNPAT mitigated CSE-induced ferroptosis. Furthermore, we found that CSE regulated the acetylation and protein levels of GNPAT by modulating SIRT4 expression. Importantly, the overexpression of GNPAT countered the inhibitory effects of SIRT4 on ferroptosis. CONCLUSIONS: Our study revealed GNPAT could be deacetylated by SIRT4, providing novel insights into the mechanisms underlying the relationship between CSE-induced ferroptosis and COPD.

Lipocalin 2 regulates mitochondrial phospholipidome remodeling, dynamics, and function in brown adipose tissue in male mice.

Mitochondrial function is vital for energy metabolism in thermogenic adipocytes. Impaired mitochondrial bioenergetics in brown adipocytes are linked to disrupted thermogenesis and energy balance in obesity and aging. Phospholipid cardiolipin (CL) and phosphatidic acid (PA) jointly regulate mitochondrial membrane architecture and dynamics, with mitochondria-associated endoplasmic reticulum membranes (MAMs) serving as the platform for phospholipid biosynthesis and metabolism. However, little is known about the regulators of MAM phospholipid metabolism and their connection to mitochondrial function. We discover that LCN2 is a PA binding protein recruited to the MAM during inflammation and metabolic stimulation. Lcn2 deficiency disrupts mitochondrial fusion-fission balance and alters the acyl-chain composition of mitochondrial phospholipids in brown adipose tissue (BAT) of male mice. Lcn2 KO male mice exhibit an increase in the levels of CLs containing long-chain polyunsaturated fatty acids (LC-PUFA), a decrease in CLs containing monounsaturated fatty acids, resulting in mitochondrial dysfunction. This dysfunction triggers compensatory activation of peroxisomal function and the biosynthesis of LC-PUFA-containing plasmalogens in BAT. Additionally, Lcn2 deficiency alters PA production, correlating with changes in PA-regulated phospholipid-metabolizing enzymes and the mTOR signaling pathway. In conclusion, LCN2 plays a critical role in the acyl-chain remodeling of phospholipids and mitochondrial bioenergetics by regulating PA production and its function in activating signaling pathways.

Ether-lipids and cellular signaling: A differential role of alkyl- and alkenyl-ether-lipids?

Ether-lipids (EL) are specific lipids bearing a characteristic sn-1 ether bond. Depending on the ether or vinyl-ether nature of this bond, they are present as alkyl- or alkenyl-EL, respectively. Among EL, alkenyl-EL, also referred as plasmalogens in the literature, attract most of the scientific interest as they are the predominant EL species in eukaryotic cells, thus less is known about alkyl-EL. EL have been implicated in various signaling pathways and alterations in their quantity are frequently observed in pathologies such as neurodegenerative and cardiovascular diseases or cancer. However, it remains unknown whether both alkyl- and alkenyl-EL play the same roles in these processes. This review summarizes the roles and mechanisms of action of EL in cellular signaling and tries to discriminate between alkyl- and alkenyl-EL. We also focus on the involvement of EL-mediated alterations of cellular signaling in diseases and discuss the potential interest for EL in therapy.

Fetal sex differences in placental LCPUFA ether and plasmalogen phosphatidylethanolamine and phosphatidylcholine contents in pregnancies complicated by obesity.

BACKGROUND: We have previously reported that maternal obesity reduces placental transport capacity for lysophosphatidylcholine-docosahexaenoic acid (LPC-DHA), a preferred form for transfer of DHA (omega 3) to the fetal brain, but only in male fetuses. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC), have either sn-1 ester, ether or vinyl ether (plasmalogen) linkages to primarily unsaturated and monounsaturated fatty acids and DHA or arachidonic acid (ARA, omega 6) in the sn-2 position. Whether ether and plasmalogen PC and PE metabolism in placenta impacts transfer to the fetus is unexplored. We hypothesized that ether and plasmalogen PC and PE containing DHA and ARA are reduced in maternal-fetal unit in pregnancies complicated by obesity and these differences are dependent on fetal sex. METHODS: In maternal, umbilical cord plasma and placentas from obese women (11 female/5 male infants) and normal weight women (9 female/7 male infants), all PC and PE species containing DHA and ARA were analyzed by LC-MS/MS. Placental protein expression of enzymes involved in phospholipid synthesis, were determined by immunoblotting. All variables were compared between control vs obese groups and separated by fetal sex, in each sample using the Benjamini-Hochberg false discovery rate adjustment to account for multiple testing. RESULTS: Levels of ester PC containing DHA and ARA were profoundly reduced by 60-92% in male placentas of obese mothers, while levels of ether and plasmalogen PE containing DHA and ARA were decreased by 51-84% in female placentas. PLA2G4C abundance was lower in male placentas and LPCAT4 abundance was lower solely in females in obesity. In umbilical cord, levels of ester, ether and plasmalogen PC and PE with DHA were reduced by 43-61% in male, but not female, fetuses of obese mothers. CONCLUSIONS: We found a fetal sex effect in placental PE and PC ester, ether and plasmalogen PE and PC containing DHA in response to maternal obesity which appears to reflect an ability of female placentas to adapt to maintain optimal fetal DHA transfer in maternal obesity.

Docosahexaenoic acid (DHA) is a critical omega 3 long chain polyunsaturated fatty acid (LCPUFA) for fetal brain development. We have recently reported that maternal obesity reduces placental transport capacity for LysophosPhatidylCholine-DHA (LPC-DHA), a preferred form for transfer of DHA to the fetal brain, but only in male fetuses. Other important lipids, the plasmalogen phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are considered DHA reservoirs, but its roles in the maternal­fetal unit are largely unexplored. We examined these lipid species in maternal and fetal circulation and in placental tissue to uncover potential novel roles for ether and plasmalogen lipids in the regulation of placenta delivery of these vital nutrients in pregnancies complicated by obesity depending of fetal sex. We demonstrated for the first time, that female fetuses of obese mothers decrease placental ether and plasmalogen PE containing DHA and arachidonic acid (ARA, omega 6), and show a high fetal­placental adaptability and placental reserve capacity that can maintain the PC-LCPUFA synthesis and the transfer of these crucial species to the fetus to preserve brain development. Our study also demonstrated that male fetuses, in response to maternal obesity, reduce the placental ester PC species containing DHA and ARA and reduce the ether and plasmalogen PE reservoir of DHA and ARA in fetal circulation. Our findings support a fetal sex effect in placental ester, ether and plasmalogen PE and PC containing DHA in response to maternal obesity which appears to reflect an ability of female placentas to adapt to maintain optimal fetal DHA transfer in maternal obesity.

Effects of high-sugar, high-cholesterol, and high-fat diet on phospholipid profile of mouse tissues with a focus on the mechanism of plasmalogen synthesis.

High-sugar diet (HSD), high-cholesterol diet (HCD), and high-fat diet (HFD) all modulate the levels of lipids. However, there is a lack of comparative data on the effects of different diets on phospholipids (PLs). Given their important role in physiology and disease, there has been an increasing focus on altered PLs in liver and brain disorders. This study aims to determine the effects of HSD, HCD, and HFD for 14-week feeding on the PL profile of the mouse liver and hippocampus. Quantitative analysis of 116 and 113 PL molecular species in liver and hippocampus tissues revealed that the HSD, HCD, and HFD significantly affected the PLs in liver and hippocampus, especially decreased the levels of plasmenylethanolamine (pPE) and phosphatidylethanolamine (PE). Overall, the impact of HFD on liver PLs was more significant, consistent with the morphological changes in the liver. Compared to HSD and HCD, HFD induced a significant decrease in PC (P-16:0/18:1) and an increase in LPE (18:0) and LPE (18:1) in liver. In the liver of mice fed with different diets, the expression of the key enzymes Gnpat, Agps in the pPE biosynthesis pathway and peroxisome-associated membrane proteins pex14p were decreased. In addition, all diets significantly reduced the expression of Gnpat, pex7p, and pex16p in hippocampus tissue. In conclusion, HSD, HCD, and HFD enhanced lipid accumulation in the liver, led to liver injury, significantly affected the liver and hippocampus PLs, and decreased the expression of genes related to plasmalogen synthesis in mouse liver and hippocampus, which caused severe plasmalogen reduction.

1-O-alkyl-glycerols from Squid Berryteuthis magister Reduce Inflammation and Modify Fatty Acid and Plasmalogen Metabolism in Asthma Associated with Obesity.

Asthma associated with obesity is considered the most severe phenotype and can be challenging to manage with standard medications. Marine-derived 1-O-alkyl-glycerols (AGs), as precursors for plasmalogen synthesis, have high biological activity, making them a promising substance for pharmacology. This study aimed to investigate the effect of AGs from squid Berryteuthis magister on lung function, fatty acid and plasmalogen levels, and cytokine and adipokine production in obese patients with asthma. The investigational trial included 19 patients with mild asthma associated with obesity who received 0.4 g of AGs daily for three months in addition to their standard treatment. The effects of AGs were evaluated at one and three months of treatment. The results of the study demonstrated that intake of AGs increased the FEV1 and FEV1/VC ratios, and significantly decreased the ACQ score in 17 of the 19 patients after three months of treatment. The intake of AGs increased concentration of plasmalogen and n-3 PUFA in plasma, and modified leptin/adiponectin production by adipose tissue. The supplementation of AGs decreased the plasma levels of inflammatory cytokines (TNF-α, IL-4, and IL-17a), and oxylipins (TXB2 and LTB4), suggesting an anti-inflammatory property of AGs. In conclusion, 1-O-alkyl-glycerols could be a promising dietary supplement for improving pulmonary function and reducing inflammation in obese asthma patients, and a natural source for plasmalogen synthesis. The study highlighted that the beneficial effects of AG consumption can be observed after one month of treatment, with gradual improvement after three months of supplementation.

Utilization of LC-MS/MS and Drift Tube Ion Mobility for Characterizing Intact Oxidized Arachidonate-Containing Glycerophosphatidylethanolamine.

Lipid peroxidation is a key component in the pathogenesis of numerous disease states, where the oxidative damage of lipids frequently leads to membrane dysfunction and subsequent cellular death. Glycerophosphoethanolamine (PE) is the second most abundant phospholipid found in cellular membranes and, when oxidized, has been identified as an executor of ferroptotic cell death. PE commonly exists in the plasmalogen form, where the presence of the vinyl ether bond and its enrichment in polyunsaturated fatty acids make it especially susceptible to oxidative degradation. This results in a multitude of oxidized products complicating identification and often requiring several analytical techniques for interpretation. In the present study, we outline an analytical approach for the structural characterization of intact oxidized products of arachidonate-containing diacyl and plasmalogen PE. Intact oxidized PE structures, including structural and positional isomers, were identified using complementary liquid chromatography techniques, drift tube ion mobility, and high-resolution tandem mass spectrometry. This work establishes a comprehensive method for the analysis of intact lipid peroxidation products and provides an important pathway to investigate how lipid peroxidation initially impacts glycerophospholipids and their role in redox biology.

Characterization and heterologous expression of plasmalogen synthase MeHAD from Megasphaera elsdenii.

Plasmalogens (Pls) are vinyl-ether bond-containing glycerophospholipids or glycosyl diradyl glycerols, and are of great importance in the physiological functions and stability of cell membrane. Here, we identified and characterized that the plasmalogen synthase MeHAD from anaerobic Megasphaera elsdenii was responsible for vinyl-ether bond formation. Different from the 2-hydroxyacyl-CoA dehydratase (HAD) family plasmalogen synthase PlsA-PlsR which are encoded by two genes in Clostridium perfringens, the HAD homolog (MeHAD) encoded by a single gene MELS_0169 was found in M. elsdenii. By heterologous expression of the MeHAD gene into a nonplasmalogen-producing Escherichia coli strain, the expressed MeHAD was found to be located in the cell membrane region. Plasmalogens were detected in the recombinant strain using GC-MS and LC-MS, demonstrating that MeHAD was the key enzyme for plasmalogen synthesis. Moreover, the synthesized plasmalogens could enhance the oxidative stress-resistance and osmotic pressure-resistance of the recombinant strain, probably due to the ROS scavenging and decreased membrane permeability by the plasmalogens, respectively. The four-cysteine (Cys125, Cys164, Cys445 and Cys484) site-mutant of MeHAD, which were predicted binding to the [4Fe-4S] cluster, was unable to synthesize plasmalogens, indicating that the cysteines are important for the catalytic activity of MeHAD. Our results revealed the single gene encoded plasmalogen synthase in M. elsdenii and established a recombinant E. coli strain with plasmalogen production potential.

Systematic crosstalk in plasmalogen and diacyl lipid biosynthesis for their differential yet concerted molecular functions in the cell.

Plasmalogen is a major phospholipid of mammalian cell membranes. Recently it is becoming evident that the sn-1 vinyl-ether linkage in plasmalogen, contrasting to the ester linkage in the counterpart diacyl glycerophospholipid, yields differential molecular characteristics for these lipids especially related to hydrocarbon-chain order, so as to concertedly regulate biological membrane processes. A role played by NMR in gaining information in this respect, ranging from molecular to tissue levels, draws particular attention. We note here that a broad range of enzymes in de novo synthesis pathway of plasmalogen commonly constitute that of diacyl glycerophospholipid. This fact forms the basis for systematic crosstalk that not only controls a quantitative balance between these lipids, but also senses a defect causing loss of lipid in either pathway for compensation by increase of the counterpart lipid. However, this inherent counterbalancing mechanism paradoxically amplifies imbalance in differential effects of these lipids in a diseased state on membrane processes. While sharing of enzymes has been recognized, it is now possible to overview the crosstalk with growing information for specific enzymes involved. The overview provides a fundamental clue to consider cell and tissue type-dependent schemes in regulating membrane processes by plasmalogen and diacyl glycerophospholipid in health and disease.

Differential Eye Expression of Xenopus Acyltransferase Gnpat and Its Biochemical Characterization Shed Light on Lipid-Associated Ocular Pathologies.

Purpose: Plasmalogens (Plgs) are highly abundant lipids in the retina, and their deficiency leads to severe abnormalities during eye development. The first acylation step in the synthesis of Plgs is catalyzed by the enzyme glyceronephosphate O-acyltransferase (GNPAT), which is also known as dihydroxyacetone phosphate-acyltransferase (EC 2.3.1.42). GNPAT deficiency produces rhizomelic chondrodysplasia punctata type 2, a genetic disorder associated with developmental ocular defects. Despite the relevance of retinal Plgs, our knowledge of the mechanisms that regulate their synthesis, and the role of GNPAT during eye development is limited. Methods: Using the Xenopus laevis model organism, we characterized by in situ hybridization the expression pattern of gnpat and compared it to glycerol 3-phosphate acyltransferase mitochondrial (gpam or gpat1) during eye neurogenesis, lamination, and morphogenesis. The Xenopus Gnpat was biochemically characterized in a heterologous expression system in yeast. Results: During development, gnpat is expressed in proliferative cells of the retina and lens, and post-embryogenesis in proliferative cells of the ciliary marginal zone and lens epithelium. In contrast, gpam expression is mainly restricted to photoreceptors. Xenopus Gnpat expressed in yeast is present in both soluble and membrane fractions, but only the membrane-bound enzyme displays activity. The amino terminal of Gnpat, conserved in humans, shows lipid binding capacity that is enhanced by phosphatidic acid. Conclusions: Enzymes involved in the Plgs and glycerophospholipid biosynthetic pathways are differentially expressed during eye morphogenesis. The gnpat expression pattern and the molecular determinants regulating Gnpat activity advance our knowledge of this enzyme, contributing to our understanding of the retinal pathophysiology associated with GNPAT deficiency.