Evidence for enzymatic backbone methylation of the main membrane lipids in the archaeon Methanomassiliicoccus luminyensis

Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers (BDGTs and PDGTs, respectively) are recently identified classes of archaeal membrane lipids that are prominent constituents in anoxic subseafloor sediments. These lipids are intriguing as they possess unusual backbones with four or five carbon atoms instead of the canonical three-carbon glycerol backbone. In this study, we examined the biosynthesis of BDGTs and PDGTs by the methanogen Methanomassiliicoccus luminyensis , the only available isolate known to produce these compounds, via stable isotope labeling with [ methyl - 13 C] methionine followed by mass spectrometry analysis. We show that their biosynthesis proceeds from transfer(s) of the terminal methyl group of methionine to the more common archaeal membrane lipids, i.e., glycerol dibiphytanyl glycerol tetraethers (GDGTs). As this methylation targets a methylene group, a radical mechanism involving a radical S-adenosylmethionine (SAM) enzyme is probable. Over the course of the incubation, the abundance of PDGTs relative to BDGTs, expressed as backbone methylation index, increased, implying that backbone methylation may be related to the growth shift to stationary conditions, possibly due to limited energy and/or substrate availability. The increase of the backbone methylation index with increasing sediment age in a sample set from the Mediterranean Sea adds support for such a relationship. Importance Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers are membrane lipids recently discovered in anoxic environments. These lipids differ from typical membrane-spanning tetraether lipids because they possess a non-glycerol backbone. The biosynthetic pathway and physiological role of these unique lipids are currently unknown. Here, we show that in the strain Methanomassiliicoccus luminyensis these lipids are the result of methyl transfer(s) from a S-adenosyl methionine (SAM) intermediate. We observed a relative increase of the doubly methylated compound, pentanetriol dibiphytanyl glycerol tetraether, in the stationary phase of M. luminyensis as well as in the subseafloor of the Mediterranean Sea and thus introduced a backbone methylation index, which could be used to further explore microbial activity in natural settings.

Isomeric lipid signatures reveal compartmentalised fatty acid metabolism in cancer

Cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular fatty acids (FA) and de novo synthesis. Given that oxidation of de novo synthesised FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates - however hitherto FAs were considered part of a common pool. To probe FA metabolic partitioning, cancer cells were supplemented with stable-isotope labelled FAs. Structural analysis of the resulting glycerophospholipids revealed that labelled FAs from uptake were largely incorporated to canonical (sn-)positions on the glycerol backbone. Surprisingly, labelled FA uptake disrupted canonical isomer patterns of the unlabelled lipidome and induced repartitioning of n-3 and n-6 polyunsaturated-FAs into glycerophospholipid classes. These structural changes evidence differences in the metabolic fate of FAs derived from uptake or de novo sources and demonstrate unique signalling and remodelling behaviours usually hidden to conventional lipidomics.

A Novel qNMR Application for the Quantification of Vegetable Oils Used as Adulterants in Essential Oils

Essential oils (EOs) are more and more frequently adulterated due to their wide usage and large profit, for this reason accurate and precise authentication techniques are essential. This work aims at the application of qNMR as a versatile tool for the quantification of vegetable oils potentially usable as adulterants or diluents in EOs. This approach is based on the quantification of both 1H and 13C glycerol backbone signals, which are actually present in each vegetable oil containing triglycerides. For the validation, binary mixtures of rosemary EO and corn oil (0.8–50%) were prepared. To verify the general feasibility of this technique, other different mixtures including lavender, citronella, orange and peanut, almond, sunflower, and soy seed oils were analyzed. The results showed that the efficacy of this approach does not depend on the specific combination of EO and vegetable oil, ensuring its versatility. The method was able to determine the adulterant, with a mean accuracy of 91.81 and 89.77% for calculations made on 1H and 13C spectra, respectively. The high precision and accuracy here observed, make 1H-qNMR competitive with other well-established techniques. Considering the current importance of quality control of EOs to avoid fraudulent practices, this work can be considered pioneering and promising.

Structure and Interdigitation of Chain-Asymmetric Phosphatidylcholines and Milk Sphingomyelin in the Fluid Phase

We addressed the frequent occurrence of mixed-chain lipids in biological membranes and their impact on membrane structure by studying several chain-asymmetric phosphatidylcholines and the highly asymmetric milk sphingomyelin. Specifically, we report trans-membrane structures of the corresponding fluid lamellar phases using small-angle X-ray and neutron scattering, which were jointly analyzed in terms of a membrane composition-specific model, including a headgroup hydration shell. Focusing on terminal methyl groups at the bilayer center, we found a linear relation between hydrocarbon chain length mismatch and the methyl-overlap for phosphatidylcholines, and a non-negligible impact of the glycerol backbone-tilting, letting the sn1-chain penetrate deeper into the opposing leaflet by half a CH2 group. That is, penetration-depth differences due to the ester-linked hydrocarbons at the glycerol backbone, previously reported for gel phase structures, also extend to the more relevant physiological fluid phase, but are significantly reduced. Moreover, milk sphingomyelin was found to follow the same linear relationship suggesting a similar tilt of the sphingosine backbone. Complementarily performed molecular dynamics simulations revealed that there is always a part of the lipid tails bending back, even if there is a high interdigitation with the opposing chains. The extent of this back-bending was similar to that in chain symmetric bilayers. For both cases of adaptation to chain length mismatch, chain-asymmetry has a large impact on hydrocarbon chain ordering, inducing disorder in the longer of the two hydrocarbons.

iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4

Here, we identify iPLA2β as a critical regulator for p53-driven ferroptosis upon reactive oxygen species (ROS)-induced stress. The calcium-independent phospholipase iPLA2β is known to cleave acyl tails from the glycerol backbone of lipids and release oxidized fatty acids from phospholipids. We found that iPLA2β-mediated detoxification of peroxidized lipids is sufficient to suppress p53-driven ferroptosis upon ROS-induced stress, even in GPX4-null cells. Moreover, iPLA2β is overexpressed in human cancers; inhibition of endogenous iPLA2β sensitizes tumor cells to p53-driven ferroptosis and promotes p53-dependent tumor suppression in xenograft mouse models. These results demonstrate that iPLA2β acts as a major ferroptosis repressor in a GPX4-independent manner. Notably, unlike GPX4, loss of iPLA2β has no obvious effect on normal development or cell viability in normal tissues but iPLA2β plays an essential role in regulating ferroptosis upon ROS-induced stress. Thus, our study suggests that iPLA2β is a promising therapeutic target for activating ferroptosis-mediated tumor suppression without serious toxicity concerns.

Phospholipid dynamics in ex vivo lung cancer and normal lung explants

In cancer cells, metabolic pathways are reprogrammed to promote cell proliferation and growth. While the rewiring of central biosynthetic pathways is being extensively studied, the dynamics of phospholipids in cancer cells are still poorly understood. In our study, we sought to evaluate de novo biosynthesis of glycerophospholipids (GPLs) in ex vivo lung cancer explants and corresponding normal lung tissue from six patients by utilizing a stable isotopic labeling approach. Incorporation of fully 13C-labeled glucose into the backbone of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinositol (PI) was analyzed by liquid chromatography/mass spectrometry. Lung cancer tissue showed significantly elevated isotopic enrichment within the glycerol backbone of PE, normalized to its incorporation into PI, compared to that in normal lung tissue; however, the size of the PE pool normalized to the size of the PI pool was smaller in tumor tissue. These findings indicate enhanced PE turnover in lung cancer tissue. Elevated biosynthesis of PE in lung cancer tissue was supported by enhanced expression of the PE biosynthesis genes ETNK2 and EPT1 and decreased expression of the PC and PI biosynthesis genes CHPT1 and CDS2, respectively, in different subtypes of lung cancer in publicly available datasets. Our study demonstrates that incorporation of glucose-derived carbons into the glycerol backbone of GPLs can be monitored to study phospholipid dynamics in tumor explants and shows that PE turnover is elevated in lung cancer tissue compared to normal lung tissue.

A promiscuous archaeal cardiolipin synthase generating a variety of cardiolipins and phospholipids

Cardiolipin (DPCL) biosynthesis has barely been explored in Archaeal isoprenoid-based ether lipid membranes. Here, we identified a cardiolipin synthase (MhCls) from the mesophilic anaerobic methanogen Methanospirillum hungatei. The enzyme was overexpressed in Escherichia coli, purified, and subsequently characterized by LC-MS. MhCls utilizes two archaetidylglycerol molecules in a transesterification reaction to synthesize archaeal di-phosphate cardiolipin (aDPCL) and glycerol. The enzyme is invariant to the stereochemistry of the glycerol-backbone and the nature of the lipid tail, as it also accepts phosphatidylglycerol to generate di-phosphate cardiolipin (DPCL). Remarkably, in the presence of archaetidylglycerol and phosphatidylglycerol, MhCls formed an archaeal-bacterial hybrid di-phosphate cardiolipin (hDPCL), that so far has not been observed in nature. Due to the reversibility of the transesterification, cardiolipin can be converted back in presence of glycerol into phosphatidylglycerol. In the presence of other compounds that contain primary hydroxyl groups (e.g. alcohols, water, sugars) various natural and unique artificial phospholipid species could be synthesized, including multiple di-phosphate cardiolipin species. Moreover, MhCls could utilize a glycolipid in the presence of phosphatidylglycerol to form a glycosyl-mono-phosphate cardiolipin, emphasizing the promiscuity of this cardiolipin synthase.

A new approach for characterization of phosphatidylcholines and lysophosphatidylcholine in human plasma

Aim: Characterization of phosphatidylcholines (PCs) and lysophosphatidylcholine in human plasma using LC-IT-MSn. The characterization approach was based on trapping the eluted positive ions and applying low voltage for fragmentation to MS2 and further fragmentation of the most abundant two peaks to obtain MS3. This approach allowed linking the MS3 data to MS2 and precursor ion. Methodology: The fatty acid part, at sn-1 and sn-2 of the glycerol backbone, could be identified based on the favored cleavage pathway. Conclusion: The dysregulated PCs and lysophosphatidylcholines in human plasma obtained from acute coronary syndrome cases, and Type 2 diabetes patients suffering no coronary syndromes were estimated and matched versus healthy volunteers. An epoxide form of 16:0–18:2 PC was confirmed, m/z 774.6.

Regiospecific Analysis of Fatty Acids and Calculation of Triglyceride Molecular Species in Marine Fish Oils

The regiospecific distribution of fatty acids (FAs) and composition of triglyceride (TAG) molecular species of fishes were analyzed and calculated by pancreatic lipase (PL) hydrolysis and Visual Basic (VB) program. DHA was preferentially located at sn-2 position in TAG molecule, whereas EPA was almost equally distributed in each position of glycerol backbone. DOP, DPP, EPP, PoPP, PPO, and PPP were the predominant TAG species. MPP in anchovy, DDP, DOP, DPP in tuna, and EOO and OOO in salmon were the characteristic TAG molecules, which were meaningful to differentiate marine fish oils. Furthermore, the data management, according to TCN and ECN, was firstly applied to classify the TAG molecular species. The ECN42, ECN46, and ECN48 groups were rich in TAGs. The lower ECN values, compared to the higher TCN values, indicated that the most abundant TAGs exhibited a higher unsaturated degree. Therefore, our study not only offered a simple and feasible approach for the analysis of TAG composition but also firstly summarized the information by data management within ECN and TCN.