Lipid Matters

5 March 2025

New phospholipid remodelling enzymes that generate fatty acyl thiamine esters

Cells dynamically remodel their phospholipid membranes to ensure that their molecular species compositions are in line with the requirements of their specific tissues. For example, immune cells contain many plasmalogens, while brain has a high proportion of longer chain n3 PUFA. Membranes are also remodelled in response to acute challenge, e.g. following agonist activation of platelets or white cells. The classic pathway for this is the Lands cycle, described by Bill Lands in the 1950s, which utilises families of enzymes from the ACSL and LPLAT/MBOAT families. These show strong cell and tissue specific expression patterns.  Over the last several decades, many of these enzymes with differing PL and FA specificities have been discovered and characterised and as they increased in number and complexity, a new nomenclature was proposed in 2022 by Shindou et al.

Recently, a new study in Science Advances from the Petkevicius lab at the MRC Mitochondrial Biology Unit in Cambridge has identified that a family of poorly understood TRAM-LAG-CLN8 domain (TLCD) containing proteins also can act as phospholipid remodelling enzymes, regulating cellular lipid composition and generating novel esters as biproducts. 

What’s also very interesting about this study is that it’s the first identification of new lipids comprised of fatty acyls attached to thiamine, which the authors proved using labelling studies, revealing palmitic, stearic and oleic-thiamine esters. These were identified in Hela cells and were dependent on expression of TLCD1 (and this was conserved in both yeast and worm).It will be interesting to see how this develops, in particular whether these novel products display unexpected biological roles.

Importantly, Sheokand et al also show that one of these proteins is a lysoPG acyltransferase, participating in lysosome function. Specifically, CLN8 is involved in generation of bis(monoacylglycero)phosphate (BMP), identifying a new way to form this family of lipids, of direct relevance to Battens disease where mutations in the protein are directly implicated.

Valerie O’Donnell, Cardiff University

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19 February 2025

Lro1 Regulates Endoplasmic Reticulum Biogenesis.

One of the ever present and interesting biological mysteries is how cellular organelles regulate their size.  In a recent study by Lysyganicz et al, the authors present some intriguing evidence regarding the role of a phospholipid diacylglycerol acyltransferases (PDAT) termed Lro1 plays a key role modulating endoplasmic reticulum biogenesis in yeast. This is interesting as we all learned that triglycerides are classically generated via the hydrolysis of phosphatic acid to diacylglycerol which is then acylated using a fatty-acyl-Coa catalyzed by diacylglycerol O-acyltransferases (DGATs). Using a catalytically depressed Lro1 variant, however, the authors provide evidence that Lro1 modulates ER membrane expansion driven by phospholipid synthesis.  Interestingly, the authors show that the subcellular distribution, and subsequent membrane turnover, of Lro1 are controlled by production of diacylglycerol via the phosphatidic acid phosphatase Pah1. The authors suggest that their data implicates a lipid-metabolic network involved in regulating endoplasmic reticulum biogenesis that involves converting phospholipids into storage lipids.

Dan M. Raben

The John Hopkins University School of Medicine, Baltimore, MD, USA

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23 January 2025

De novo lipid synthesis and polarized prenylation are involved in cell invasion

As has been known, during development, immune surveillance, and in cancer metastasis, cells must breach and clear basement membrane barriers.  It appears that large, transient, specialized lipid-rich membrane protrusions are used in this process.  One popular model system used to examine this process is the anchor cell invasion of Caenorhabditis elegans. In a study published in July, Park et al. used live imaging, endogenous protein tagging, and cell-specific RNAi show that lipogenesis and a polarized lipid prenylation drives for formation of the invasive protrusion of the anchor cells.  The SREBP-dependent expression of fatty acid synthesis enzymes POD-2 (acetyl-CoA carboxylase, ACC), FASN-1 (a fatty acid synthase), the ZMP-1 matrix metalloproteinase and the endoplasmic reticulum localized HMG-CoA reductase HMGR-1, which generates isoprenoids for polarized prenylation, are all involved.  This study highlights the importance of coordinated lipid synthesis in the formation of cellular protrusions that are essential for basement membrane invasion.

Dan M. Raben

The John Hopkins University School of Medicine, Baltimore, MD, USA

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23 December 2024

Lipid Regulation of Adenlylyl Cyclase

Most, if not all. of us learned about the regulation of membrane-bound adenylate cyclases (often referred to as mACs) during our training and careers as scientists.Typically, we learned about the regulation of these enzymes by specific G-proteins which are themselves activated by specific membrane receptors (G-protein-coupled receptors (GPCRs)). Over time, it became clear that these enzymes are regulated by a variety of mechanisms, all of which are molecules present in the cytosol. In a recent study, Landau et al (eLife 2024;13:RP101483) have discovered the transmembrane domains of mACs respond to aliphatic lipid and anadamide to control mACs stimulated by Gsα.  The lipid signals enhance some mACs isoforms, while attenuating other isoforms. Using chimeric constructs of the various isoforms of mACs, they further showed that the hexahelical transmembrane domains of these enzymes serve as receptors for these signals. Their results open up and new, and rather novel regulatory mechanism of mACs that had been unrecognized and potentially very important.

Up to now the two hexahelical transmembrane domains of mACs were considered to fix the enzyme to membranes. Here, we show that the transmembrane domains serve in addition as signal receptors and transmitters of lipid signals that control Gsα-stimulated mAC activities. We identify aliphatic fatty acids and anandamide as receptor ligands of mAC isoforms 1–7 and 9. The ligands enhance (mAC isoforms 2, 3, 7, and 9) or attenuate (isoforms 1, 4, 5, and 6) Gsα-stimulated mAC activities in vitro and in vivo. Substitution of the stimulatory membrane receptor of mAC3 by the inhibitory receptor of mAC5 results in a ligand inhibited mAC5–mAC3 chimera. Thus, we discovered a new class of membrane receptors in which two signaling modalities are at a crossing, direct tonic lipid and indirect phasic GPCR–Gsα signaling regulating the biosynthesis of cAMP.

Dan M. Raben - The John Hopkins University School of Medicine, Baltimore, MD, USA

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10 December 2024

The ever-evolving roles of PGE2.

PGE2 is one of the oldest known members of the prostaglandin family of oxygenated fatty acids. Originally discovered by Bergström and Samuelsson, it won them the Nobel Prize in 1982, along with Vane for discovery of how aspirin blocks cyclooxygenase.  Since then, our knowledge of its roles in both health and disease have continued to expand and nowadays it’s not only considered the pro-inflammatory cause of redness, pain and fever, but also has under several circumstances established itself as an important anti-inflammatory mediator.

Recently, another immunomodulatory function for PGE2 was revealed by Bohnacker et al in Science Immunology. Here, they focused on identification of how helminths, important intestinal parasites particularly in low-income countries, evade host immunity. Using a mouse model, they found that a worm enzyme glutamate dehydrogenase drives chronicity by suppressing macrophage functions. Here, the enzyme was already known to suppress allergic inflammation in asthma, but how it regulated immune evasion of helminths wasn’t known. 

An unusual mechanism was revealed, specifically the enzyme appears to become internalized into macrophages, where it regulates the TCA cycle and amino acid metabolism. Having said that, the authors point out that it’s not currently clear if internalization of the protein is required for this function or how it’s taken up by macrophages. Next, they showed that its non-catalytic N terminus upregulates expression of PGE2 synthetic enzymes including COX2, mPGES-1, and others.The PGE2 then suppresses alternative macrophage activation which allows the worm to evade immune clearance.  Although not discussed in this paper, since it was acknowledged that this specific worm does not infect humans, should this mechanism operate in human worm infection also, simple treatments targeting PGE2 and its signalling could be envisaged. 

Valerie O'Donnell, Cardiff University

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25 November 2024

PLD3 and PLD4 synthesize S,S-BMP, a key phospholipid enabling lipid degradation in lysosomes

Phospholipase D (PLD), first cloned from castor beans, is a family of six known isoforms.  PLD1 and PLD2, the most widely studied isoforms of the mammalian PLDs, are catalytically active in catalyzing the hydrolysis of its principal substrate phosphatidylcholine to phosphatidic acid and free choline.  PLD3 and PLD4 are transmembrane proteins in the endoplasmic reticulum with no known catalytic activity but thought to have non-enzymatic functions in the ER.  In a recent excellent study by Singh et al (Cell 187, 1–15, November 27, 2024) the authors present data showing PLD3 and PLD4 synthesize an intraluminal lysosomal lipid, bis(monoacylglycero)phosphate vis the transphosphatidylation of lyso-phosphatidylglycerol, with the unusual stereochemical configuration where both glycerol carbons are in the S configuration (S,S-BMP). This prevents this lipid from being degraded by lysosomal phospholipases.Interestingly, loss of PLD3 and PLD4 not only leads to the absence of S,S-BMP synthesis, it leads to the accumulation of gangliosides and other lysosomal abnormalities. The authors suggest that S,S-BMP mediates the degradation of gangliosides. As PLD3 and PLD4 are exonucleases, the authors suggest that PLD3 and PLD4, like mitochondrial PLD6, may act as both lipases and exonucleases. This study has opened up some interesting and potentially important aspects of PLD research.

Daniel M. Raben - The John Hopkins University School of Medicine, Baltimore, MD, USA

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11 November 2024

The impact of viral infection on the lipidome continues to be a topic of high interest to researchers. Of course, it’s long been known that enveloped viruses in particular hijack the host lipidome in order to generate sufficient membrane to support replication. What’s been less clear is how this is achieved, in particular what specific genes are required and how this impacts the lipidomes of the host cells.

This week, focusing on several orthoflavivirus strains, Herner et al demonstrate that glycerophospholipid (PL) remodelling is essential for replication in vitro (Glycerophospholipid remodeling is critical for orthoflavivirus infection). Early changes included increased TAG and CE, as well as lysoPL, while PL such as PE, PS and PC were all reduced. Next, the study made use of BioPAN, a tool released by LIPID MAPS a number of years ago that uses lipidomics to suggest genes that maybe responsible for changes in lipid levels in a cellular or tissue system (BioPAN: a web-based tool to explore mammalian lipidome metabolic pathways on LIPID MAPS). Highlighting the complexity of predicting gene changes using lipidomics, an area that’s still in its infancy, it’s important to remember that tools such as these don’t account for post-translational modifications so while they may predict which enzyme activity is up or down, this doesn’t necessarily mean the change is at the transcriptional level (versus changes in substrate supply, enzyme phosphorylation or other mechanisms for activating enzymes like calcium signalling).  In gene transcription data from the host cells didn’t (apart from PLA2G4C) match those predicted in silico.  Taking this into account, the mechanisms for the changes in PL levels need further exploration, since simple changes in gene expression don’t seem to provide a clear answer.

Next, the new study found that genetic deletion of various enzymes involved in the biosynthesis of phosphatidylserine and phosphatidylinositol reduce virus replication while blockade of ceramide biosynthesis had variable effects on titre levels and cell death depending on which enzyme was deleted.  These new data reveal several gene targets that are directly anti-viral, and although the study focused on orthoflaviviruses, which include Zika, West Nile, dengue and yellow fever, these findings maybe also relevant for respiratory enveloped viruses such as influenza and SARS/MERS viruses.  

Valerie O'Donnell, Cardiff University

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28 October 2024

Inhibition of PSS1 promotes LDL uptake via increase in LDL receptors

In mammalian cells, the synthesis of phosphatidylserine (PS) is catalyzed by two calcium-dependent, apparently mitochondrially membrane associated, PS synthases (PSS). These enzymes catalyze the exchange of serine with the choline head group of phosphatidylcholine (PC), designated PSS1, or the ethanolamine head group of phosphatidylethanolamine, designated PSS2.  While there is evidence suggesting some functional redundancies between PSS1 and PSS2, gain-of-function mutations in the gene coding for PSS1 lead to aberrant increased PS production in the endoplasmic reticulum (ER) which is believed to be involved in the pathogenesis of Lenz-Majewski syndrome (LMS).  These mutations have also been shown to alter the metabolism of membrane lipids including the transport of cholesterol out of the ER. A recent report by Long et al. examines the cryo-EM structures of wild-type human PSS1 (PSS1^WT), the LMS-causing Pro269Ser mutant (PSS1^P269S), and PSS1^WT in complex with its inhibitor DS55980254.  Interestingly, these structures suggest a mechanism of PSS1 that is related to the postulated mechanisms of the membrane-bound O-acyltransferases.  Importantly, the data indicates that both PS and DS55980254 allosterically inhibit PSS1 and that inhibition by DS55980254 activates the SREBP pathways enhancing the expression of LDL receptors leading to increased cellular LDL uptake.  Overall, these data propose a mechanism of mammalian PS synthases and suggest that selective PSS1 inhibitors could lead to lower blood cholesterol levels.

Daniel M. Raben - The John Hopkins University School of Medicine, Baltimore, MD, USA

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14 October 2024

This week’s blog is about the recent plasma ceramide ring trial, led by Federico Torta, Markus Wenk and others from the Singapore Lipidomics Incubator.  My own lab was fortunate enough to be one of the 34 participating groups and the full report of this trial was this month reported in Nature Communications: Torta et al, Concordant inter-laboratory derived concentrations of ceramides in human plasma reference materials via authentic standards. 

So, why is a ceramide plasma ring trial an important endeavor? As it stands, not many lipid categories are routinely measured in diagnostic laboratories using mass spectrometry. Partly this is down to the specialist nature and relative high cost of lipidomics, but also the fact that there needs to be a clearly defined clinical utility for the assay.  In the case of ceramides, interest in measuring these lipids has evolved over the last 20-30 years, since Yusuf Hannun and colleagues discovered their role in cancer cell death, along with characterization of their numerous and diverse structures. More recently, it was found and then replicated widely, that certain ceramides could predict risk of a vascular event. This observation led to a test being developed and licensed for cardiovascular risk clinically although as yet, this test isn’t widely adopted for testing risk in patients and it’s not yet fully established if (or how) altering ceramides will reduce event incidence.  A comprehensive news item by Mitch Leslie published last year in Science summarizes all this in more detail: Straight from the Heart.

So, considering all this, a ring trial for ceramides seems to have come along at the right time. As Torta and colleagues wrote “The main goals … were to: (i) evaluate the degree of accuracy and concordance obtained in a large inter-laboratory trial using the same shared samples and custom-tailored calibrant materials; (ii) highlight technical issues contributing to variability and technical outliers to avoid in future; (iii) document as precisely as possible the absolute concentrations of four circulating lipids in a publicly available standard reference material and (iv) lay the foundation for determination of MS-based lipidomic RI in diverse human populations across the world in a standardized fashion.”  

Importantly, not all labs used the same assay. Some used their own established methods and others, like ours, set up the Singapore SOP, which was already well standardized, and all analyzed the same plasma reference material. In summary, the study defined analytical variability and made several recommendations around use of shared reference material and authentic labelled standards, pushing the clinical analysis of ceramides one step further to routine use for cardiovascular risk testing in humans.    

Valerie O'Donnell, Cardiff University

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30 September 2024

Etomoxir Appears to Lack Assumed Specificity

In full disclosure, I’m posting an article published by a colleague, but I believe it is worth informing the community of lipid scientists of this finding. The article, titled “Etomoxir repurposed as a promiscuous fatty acid mimetic chemoproteomic probe” (Choi et al, iScience Volume 27, Issue 9, 20 September 2024) focuses on assessing the specificity of drug often used to inhibit carnitine palmitoyltransferase I, Cpt1, in order to block mitochondrial fatty acid β-oxidation.  The authors probed the specificity of etomoxir by using click enabled etomoxir probes.  While the click-etomoxir retained its inIn hibitory effect on fatty acid oxidation, it labeled numerous proteins in cells in vitro and in vivo. Many of the identified proteins were involved in the transport and metabolism of fatty acids in the cytoplasm, peroxisome, and mitochondria. Interestingly, by using promiscuous, covalent, and fatty acid mimetic properties of etomoxir, etomoxir targets of fatty acid ω-oxidation were revealed following the loss of the peroxisomal protein Pex5. The study clearly demonstrates that etomoxir is not specific for Cpt1 as previously asserted and shows that much care should be employed when using this tool to distinguish the biological effects of fatty acid oxidation.

Daniel M. Raben

The John Hopkins University School of Medicine, Baltimore, MD, USA

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16 September 2024

Last week was a busy one for LIPID MAPS when we hosted around 40 visitors from around the world for our business meeting at Cardiff, covering nomenclature, classification, software, tools, resources and education. The last time we had an in person meeting was 2019 at Babraham, following which we were interrupted by the pandemic. The meeting hosted a diverse group, which included lipid biochemists, analytical chemists, databasing and informatics experts, members of our strategic advisory board, and collaborative partners. We focused on reviewing our work over the last number of years, scoping out new projects and networking. 

As part of presenting on the background, I found a slide from 2019 which showed where the field of lipid research was 5 yrs ago. Issues considered important at that time included the changing demographic of lipid researchers from biochemistry to larger scale lipidomics, including the emergence of untargeted profiling and its associated annotation challenges, the need for high quality targeted methods for clinical applications, emerging technologies that might be on the horizon and challenges in supporting systems biology of lipids.  So where are we now?

Training and support for researchers, including provision of workshops and guidelines has been a big focus for many of us, including International Lipidomics Society, EpiLipidNET and LIPID MAPS, and this continues. A major guideline and associated checklist was published by ILS (ILS Journal). Relating to targeted methods, several ring trials were published, including the most recent by Torta et al on ceramides which is in press in Nat Comms. The biggest jump in MS technologies occurred in the area of enhanced fragmentation, for example, UV photodissociation, oxone-induced dissociation or other methods which allow lipids to be analysed using LC, then fragmented bond by bond to allow assignment of double bond and Sn positions. This is going to lead to the identification of many new lipids which will need to be curated. Here is an example from Michael et al. Challenges supporting systems integration of data are still very present and were a topic of active discussion, and related to that, the use of AI for several aspects of our work at LIPID MAPS was briefly covered.  

Many new ideas for supporting lipidomics came from the meeting and now the challenge will be in deciding which to prioritise and also, how to fund them through grant applications and new partnerships. 

Valerie O’Donnell

Cardiff University

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6 September 2024

High expression of oleoyl-ACP hydrolase underpins life-threatening respiratory viral diseases

It is well known that lipids play an important role in respiratory physiology (think surfactant). A recent study by Jia et al has added another lipid, oleic acid, in the replication of influenza virus and severity of disease (Jia et al). The investigators examined patients hospitalized with avian A(H7N9) influenza to identify early drivers of fatal disease. In a transcriptomics study they found that oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating the release of oleic acid from the oleyl-acyl-carrier protein, was strongly linked to fatal A(H7N9) disease. Interestingly, high OLAH levels were correlated with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C).  Using OLAH knockout (olah^-/-) mice, a lethal dose of influenza virus led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation.  The investigators further showed that the inhibition of lipid droplet formation led to reduced viral infection in macrophages and supplementation of oleic acid increased influenza virus infection in macrophages and inflammation.  The authors suggest these data provide mechanistic insights into how the expression of OLAH drives life-threatening respiratory disease.

Daniel M. Raben

The John Hopkins University School of Medicine, Baltimore, MD, USA

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22 August 2024

Keep the Tooth Fairy at bay… lipids and tooth decay

While it may be world tooth fairy day, tooth loss or a visit due to tooth decay may not be something you want to encourage.

The negative links between oral health and lipid profile shows another reason to brush your teeth. A nationwide cohort study, on Oral health and changes in lipid profile, in Korea, was carried out. Their finding suggest a strong link between tooth disease/loss and an individuals lipid profile being poor. It was shown that “that periodontitis and tooth loss are associated with decreased HDL cholesterol levels and increased triglyceride levels”. In contrast, the group where frequent tooth brushing (≥3 per day) occurred, there was a reduction in tooth decay/loss, it was implied, that this in turn lead to an increase in HDL cholesterol levels and decrease triglyceride levels. However, it is not stated whether the differences in lipid profile within these two groups were observed as the cause or effect, i.e. is tooth decay/loss dependant on an individual’s lipid profile or does tooth decay/loss lead to the alterations in lipid profile.

Are lipids the cause or the solution?

While there is only limited evidence for the benefits of the use of lipid to reduce tooth decay, this is what a paper by Kensche et al., Lipids in preventive dentistry propose . They suggest, that by adding hydrophobic characteristics to the tooth surfaces, in oil form, it could reduce damaging bacterial colonization and ultimately decrease the tooth susceptibility to disease. They also suggested that the use of lipids could allow the teeth to become more resistant to acid exposure and thus reduce erosion of the enamel, again protecting the teeth.

Their conclusions was that “edible oils” may be used as a preventative measure to stop erosion, and periodontal diseases.

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19 August 2024

To those of us unfamiliar with the biochemistry of amphibians, the sterol compounds bufadienolides have a very unusual structure. They are bile acid-like compounds but the regular five-carbon side-chain is cyclised into a lactone containing two carbon-carbon double bonds to give a pyran-2-one ring. Bufadienolides are secreted by toads to provide a defence mechanism against predators and parasites.

In a recent paper Chen and colleagues (J. Agric. Food Chem. 2024, 72, 17377−17391) describe the isolation and structural determination of a series of bufadienolides esterified to fatty acids at C-3 of the steroid A-ring from fertilised toad eggs. Chen et al used classical liquid extraction and column methods to isolate these molecules and a combination of spectroscopic methods for structural determination. Some of the conjugates have significant biological activities. Compounds with an aldehyde group at C-19 and β-hydroxyl groups at C-5 and C-14 having potent and broad spectrum antiproliferative effects. This work extends the diversity of bile acid derivatives and adds to the list of biological properties of this family of molecules.

Bill Griffiths

Swansea University

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6 August 2024

Membrane Localization of Proteins And Rates of Cellular Signaling

Those of us interested in membrane biology and biochemistry often wonder about the efficiency of protein-protein interactions at the membrane interface. It has been suggested that the localization of proteins to this interface will increase intermolecular association rates. This is often met with skepticism as it has been widely believed this association would be slower at the membrane than those rates in the cytosol. This question is important as many signaling events depend on the translocation of essential components which then must associate with other translocated proteins or membrane resident proteins. In a recent paper, Huang et al (Proc. Natl. Acad. Sci. Mar 5;121(10), 2024) addressed this directly by comparing the binding of complementary DNA strands, in solution and on supported membranes. Surprisingly, they discovered that rate constants within a 10µm radius spherical cell the association is 22-33-fold faster at the membrane than in the cytoplasm. They also point out, however, that this kinetic advantage depends on cell size and is essentially negligible for typical ~1µm prokaryotic cells. It seems, therefore, that while the rate constants are significantly affected at typical prokaryotic cell membranes, they are not slower either. The rate enhancement observed in smaller regions is believed to be attributable to both higher encounter rates at the membrane and an increase in reaction probability per encounter. This may be important when considering interaction and reaction rates when proteins are targeted to specific, and restricted membrane regions.

Daniel M. Raben,

The John Hopkins University School of Medicine, Baltimore, MD, USA

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28 July 2024

Hepatitis and lipids 

28 July is World Hepatitis Day – 'The date of 28 July was chosen because it is the birthday of Nobel-prize winning scientist Dr Baruch Blumberg, who discovered hepatitis B virus (HBV) and developed a diagnostic test and vaccine for the virus' (WHO). Affecting over ~350million people worldwide, mainly in Asia, Hepatitis B virus (HBV) poses a major public health problem. While HBV vaccines and effective antiviral drugs have been available, up to 90% of HBV-infected infants and children, and around 5% of HBV-infected adults develop chronic HBV infection (1).

Hepatitis B virus (HBV) and it’s effects on lipid metabolism may be unknown, however a recent review by Zhang et al. suggested “potential targets to inhibit HBV replication or expression by decreasing or enhancing certain lipid metabolism-related proteins or metabolites”. By altering both lipid synthesis and lipolysis, HBV stimulates many changes in hepatic lipid metabolism. Given these changes, the article suggests that targeting certain lipid metabolism pathways could be a potential therapeutic way to chronic hepatitis B. While further studies are needed this review could begin to solve this crisis. 

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9 July 2024

Aquaporin-0 Array Formation and Lipid Rafts?

There is a wealth of biological membranes that have received considerable attention from membrane biologists and biochemists.  Understanding the composition and biophysical properties of membranes leads to insights regarding their impacts on membrane proteins as well as membrane biochemistry and functions.  While we are learning a lot about the membranes that surround a variety of mammalian cells and their internal organelles, much less attention has been given to the membrane that encapsulates the ocular lens (lens membranes).  There are two interesting properties of lens membranes.  First, they contain a high percentage of sphingomyelin and cholesterol.  Second, the major protein in these membranes is a specific water channel designated as aquaporin-0 (AQP0).  Interestingly, AQP0 tetramers form large 2D square arrays (crystals) but the mechanism underlying the formation of this structure has not received much attention.  A recent paper by Chiu et al (https://elifesciences.org/reviewed-preprints/90851) has addressed this issue.  In this manuscript the authors present electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and used molecular dynamics (MD) simulations to identify some interesting properties.  The MD simulations show cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. The authors suggest that the properties that drive AQP0 array formation could also be responsible for protein clustering in lipid rafts opening up new insights into protein-lipid interactions and functions in membranes.

Daniel M. Raben,

The John Hopkins University School of Medicine, Baltimore, MD, USA

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24 June 2024

Last week, it was a pleasure to be part of a review panel for the German Research funding organisation Deutsche Forschungsgemeinschaft (DFG).  The programme we reviewed fell under the Priority Programmes (SPP) area and is called “Ferroptosis: from Molecular Basics to Clinical Applications”.  Like all SPP programmes, it runs for 6 years, being split into two 3 year periods, and this time, researchers across Germany were competing for the second tranche of project specific funding. This to hosted within a consortium (led by Marcus Conrad at Helmholz Zentrum München) that focuses on excellent research, collaboration, training and networking with a strong ethos of equality.  Two enjoyable days were spent hearing about the seminal discoveries made by the consortium, and the proposed follow on and new work, that covers diverse applications including cancer, neurodegeneration, and underpinning mechanisms. 

Ferroptosis is the relatively recent name given to cell death dependent on iron and lipid peroxidation.  While the premise that redox cycling by metals in concert with lipid hydroperoxides (or hydrogen peroxide, aka Fenton chemistry) can cause cell death has been around for decades, it’s only been in recent years that a specific name was assigned to it. The free radical theory of ageing was originally proposed by Denham Harman in the 1950s.  Here Fenton chemistry was proposed to be causative in inflammation, cardiovascular disease, neurodegeneration, cancer and ageing. This led to many theories about how antioxidants could prevent disease and increase longevity.  This was strongly advocated by Linus Pauling in the 1970s, who used theoretical arguments to (inaccurately) claim that extremely high doses of Vitamin C would help us “live longer and feel better”.  However, this wasn’t to be and slowly, research moved on, and the free radical theory of ageing fell by the wayside around 2014. 

Notwithstanding the history of the field, oxidative damage involving lipids and iron causes significant levels of tissue damage and cell death, and preventing this, or activating it selectively remains to be exploited therapeutically. Key to this is a more in depth understanding of the mechanisms involved.  Assigning the iron/lipid peroxidation-dependent cell death process a name is transforming research on this topic.  In particular, recent seminal work by many groups has unveiled new protein players or endogenous small molecules which either promote or prevent ferroptosis in mammalian cells, while other studies investigate the potential role of harnessing this pathway, e.g. in cancer therapy.  This new knowledge is revealing important new paradigms, as one example the recent elucidation of a role for PGE2 in regulating IL2 signaling and mitochondrial function, recently published in Nature by the SPP consortium.

A challenge for the field remains in drug development and clinical applications.  Antioxidants and metal chelators were up to now the only choice available, however they did not in the past translate to effective therapies. However, with the advent of many new protein targets, discovered in this new wave of research, that situation may to strongly poised to change.  The second tranche of the DFG SPP programme will be very interesting to watch in this regard.  

Valerie O’Donnell, Cardiff University.

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14 June 2024

How blood donation can alter the lipid profile

Not only can giving blood save the life countless others, research suggests it may also improve YOUR OWN health!!!

World Blood Donor Day a World Health Organization campaign has been celebrated on the 14th of June for the last 20 years and seeks to inform and encourage new blood donation globally. 

In this short blog I explore the health benefit claims that regular blood donation can decrease the ‘bad lipids’ in the blood. While there may be conflicting papers discussing the health benefits of donating bloody, which may include a reduction in heart rate, blood pressure and weight, here I look further into the lipid profile of blood and see what effects blood donation might be having. 

A paper published by EI Uche, Lipid profile of regular blood donors, concluded that donating blood regularly will contribute to a reduction in the LDL/HDL ratio. This ratio is used as an indicator, which when high, suggests an increased cardiovascular risk. Overall, this study showed “that regular blood donation is associated with lowering of serum lipids”. We can therefore conclude that, when regularly donating blood, there is a reduction in the LDL/HDL ratio, which could reduce the risk of developing heart disease. 

More recent studies have also concluded the same, in a paper by Kebalo AH, Lipid and Haematologic Profiling of Regular Blood Donors Revealed Health Benefits, it was suggested that regular blood donations “has a significant health benefit by lowering TC, TG and LDL-c”. When elevated, these have the potential risk of contributing to the development of chronic inflammation, therefore any reduction could decrease this overall risk.

This proposed reduction, when regularly donating blood, in cholesterol and triglyceride levels, often associated with heart disease and stroke, should certainly encourage anyone who doesn’t already donate blood to get signed up!

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10 June 2024

Essential Role for the C-terminal Domain of Oleate Hydratase

Microorganisms are known for having unique and interesting enzymes.  One class of such unique enzymes that are particularly interesting are the fatty acid hydratases.  Oleate hydratase (OhyA) is a flavoprotein which catalyzes the hydroxylation of oleic acid to 10-(R)-hydroxy stearic acid (10-HSA).  The structure and mechanism of OhyA from Staphylococcus aureus was first described in 2021 in Chuck Rock’s laboratory at St. Jude’s Research Hospital (J Biol Chem.2021;296:100252).  As an oleate hydratase, OhyA must access its substrate which is embedded in membrane bilayers.  Therefore, this enzyme must be able to extract the fatty acid from the membrane and encapsulate it within its active site.  In a recent report by Radka et al, the Rock lab provided data illuminating the critical role of the carboxy terminus in assembling the enzyme on a bilayer.  They showed that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) interacts with negatively charged phosphatidylglycerol (PG) in the bilayer. They showed that the CTD region is sufficient for membrane association with nanomolar affinity.  Interestingly, the authors showed that while the CTD binds the PG surface, it does not insert into the bilayer.  Overall, their data show that the binding of OhyA CTD to a PG surface is essential for obtaining bilayer-embedded unsaturated fatty acids.  As a side note, this article was especially difficult to write as Chuck suddenly passed away last September.  His kindness, intelligence, humanity and contributions to science will be sorely missed.  

Daniel M. Raben,

The John Hopkins University School of Medicine, Baltimore, MD, USA

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28 May 2024

Multiple Sclerosis and what lipids have the power to achieve

Ahead of World Multiple Sclerosis (MS) Day on 30th of May, my blog covers the possible role of lipids in MS diagnosis and treatment. 

MS is one of the most common diseases of the central nervous system (CNS). It is estimated around 3 people around the world have MS, with 300 being diagnosed every day.  This disease has a complex pathogenesis, which includes two main processes: immune-mediated inflammatory demyelination and progressive neurodegeneration with axonal loss. Lipids may play a crucial role in underlying immunopathogenesis of MS.

A review on ‘New Insights into Multiple Sclerosis Mechanisms’ highlights key features of lipids in this field. A key component in axonal myelin sheaths are lipids, therefore it is logical for them to play a central role in disease progression; both in inflammatory demyelination and progressive neurodegeneration. Research suggests that lipids may also be a useful diagnosis tool, when lipid biomarkers are considered. Lipidomics could also be considered when monitoring disease progression, such as lipid analysis which could demonstrate specific biomarkers for MS. At present new drugs are being designed in which lipids are both targets of treatment and carriers of novel therapeutics (see my previous blog on lipid nanoparticles). 

The review concludes that sphingolipids, phospholipids, glycerolipids, and sterols are worth further investigation in their role in MS, leading to better future diagnosis and treatment. 

Lauren Cockayne, Cardiff University

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13 May 2024

Optogenetics and Lipid Imaging Highlights the Role of Lipids In Neural Plasticity

Perhaps one of the most recognized lipid metabolizing enzymes involved in regulating membrane involved signaling and dynamics are the phospholipases C (PLCs). This has inspired numerous studies to examine the precise role of these enzymes in various physiologically important membrane processes.  These studies, however, are often limited in resolution by our inability to precisely examine the localization of these enzymes and their resulting lipid metabolism.  Recently Kim et al (Cell Chem. Biol. 31: 1-13, 2024) have taken advantage of optogenetics to examine the spatiotemporal dynamics of PLCβ membrane localization and activation on membrane lipid metabolism and neurophysiology.  These investigators used an opto-PLCβ that uses a light-induced dimer module, to direct the enzyme to the plasma membrane in a light-dependent manner.  Using phospholipase D (PLD) and diacylglycerol (DAG) kinase inhibitors, they provided evidence that a PLD contributes to DAG clearance in the membrane.  Further, the authors show that the opto-PLCβ enhances amygdala synaptic plasticity and associative fear learning in mice.  This study shows the power of using optogenetic approaches to examine the biological and physiological impacts of induced membrane lipid metabolism.

Daniel M. Raben,

The John Hopkins University School of Medicine, Baltimore, MD, USA

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30 April 2024

Today, I came across an interesting paper on new lipid scramblases published in the current issue of PNAS, but there was a paywall, and unusually, no institutional access.  It seems that only 27 UK Higher Education institutions have access to PNAS, with many Russell Group institutions (Cambridge, Manchester, Bristol, Glasgow, Birmingham) missing off the list.  However, the authors had last year deposited a preprint on BioRxiv, which is free and not for profit, so, that’s the version I’m blogging about this week (Paper: Lipid scrambling is a general feature of protein insertases).   I’d encourage everyone to use BioRxiv, MedRxiv or other preprint servers so that not only those of us in academia can read papers, but everyone can.

This new study, from Li et al in Yale and Switzerland claims that protein insertases, which are well known to translocate peptides across membranes, also act as lipid scramblases in the ER and mitochondria.   Phospholipids are generated in the ER, but only on the cytosolic side, and so to allow for membrane expansion, they need to be equilibrated by “scramblases”, which move them between both leaflets.  Up to now, only a small number of scramblases were known, and these tend to be mainly in the plasma membrane, such as those involved in apoptosis or platelet activation which work on PE and PS mainly.  However, which scramblases were involved in membrane biogenesis was totally unknown. 

In this study, Li et al postulated that a group of proteins called protein insertases could be involved, since they had similar structural features, notably a hydrophobic channel and ability to locally thin membranes.  To test this, they used liposomes which contained lipids labelled with nitrobenzoxadiazole, which allowed them to bind to BSA, but only if they were on the outer leaflet.  This led to fluorescence changes indicative of internal or external localisation.  They then reconstituted several proteins into the liposomes to determine which could scramble the membranes. Most of the tested proteins had scrambling activity, with only two showing none.  This was followed by molecular dynamics simulation studies which was validated using proteins either with or without scrambling activity.  Overall, the MDS was able to reproduce the in vitro scrambling activity.  

This interesting work revealed several new candidate lipid scramblases, but what is missing are studies in cells to validate in vitro and in silico findings.  The authors consider that this will be almost impossible since these proteins have other critical functions in cells.  It would be interesting to consider how to overcome this issue and truly reveal whether protein insertases are indeed also lipid scramblases in cells and in vivo.

Valerie O'Donnell, Cardiff University

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24 April 2024

Lipids in the world of immunization – the role of lipid nanoparticles in mRNA vaccines

Keeping my blogs topical and in line with current global events, as it is World Immunization Week I thought I would write about the relevance of lipids in vaccines.

While mRNA nor indeed the idea of mRNA vaccines is not new, the approval and use of lipid nanoparticles (LNPs) for the delivery of mRNA vaccines is innovate and exciting (1).   LNPs hit the headlines when the COVID-19 mRNA vaccine was approved for use in December 2020. The mRNA vaccines, produced by Pfizer and BioNTech, quickly followed by Moderna were the first mRNA vaccines authorized for clinical use. 

The Nature review  “Lasting impact of lipid nanoparticles”, delves further into the background, that demonstrates that without lipid nanoparticles, the mRNA COVID-19 vaccine would not have been possible. In essence, LNPs, which are generally made up of four different lipids, comprising cationic or ionizable lipids, phospholipids, cholesterol and polyethylene glycol functionalized lipids (PEG-lipids) (1), provide a protective ‘wrapper’ to transport and deliver the mRNA into cells. Crazy to think how these lipids have transformed drug delivery!!

Find out more about World Immunization Week on the WHO webpage. 

Lauren Cockayne 

Cardiff University 

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15 April 2024

Lipids Tune Membrane Mechanical Properties for Fusion

Membrane fusion is a common occurrence in a wide variety of biological processes.  There have been studies showing particular lipid and their relative compositions in membranes can have profound effects on the fusing potential of membranes.  Indeed, studies have pointed to the fact that the inner leaflet of the plasma membrane is more fusogenic than the outer leaflet.  In a fascinating recent study by Lira et al (J. Biol. Chem 299(2); 105430, 2023) the authors use the fusion of large unilamellar vesicles and giant unilamellar vesicles and a combination of confocal microscopy and time-resolved imaging to uncover the mechanical membrane properties that regulate membrane fusion.  Their data show how lipids affect membrane mechanics that modulate membrane fusion and the resulting post-fusion stability.  Interestingly, their data suggest that cholesterol somewhat reduces fusion efficiency and pore formation, it also has an indirect role in establishing a competent environment for fusion proteins.

Daniel M. Raben,

The John Hopkins University School of Medicine, Baltimore, MD, USA

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2 April 2024

High quality software is essential for both analysing and then managing the massive lipidomics datasets that are generated from today’s mass spectrometry experiments. One salient example is where fragmentation of every single detected molecule is undertaken, followed by “identification” of as many lipids as possible, using databases containing information about putative product ions. 

Considering we simply don’t have MS/MS data on every single molecule in every category, reference libraries based on in silico data are increasingly applied to identification of both knowns and unknowns. Up to know, strategies to generate these libraries mainly included rule-based and combinatorial fragmentation approaches. However, these don’t take into account gas phase chemistry that occurs during collision induced dissociation, and more recent attempts to generate better prediction including quantum-chemical computation are being developed.

In the meantime, where does that leave us in relation to trustworthiness of identifications obtained using existing libraries?  To test this out, Tetering and Oomens conducted a spectroscopic test, and on the basis of their findings, suggest that fragment ion structure annotation in MS/MS libraries are “frequently incorrect” (van Tetering, L., Spies, S., Wildeman, Q.D.K. et al, 2024).   Here, they used infrared ion spectroscopy to characterise individual product ions and compare them with those proposed by HMDB, METLIN and mzCloud. In some cases, the structure wasn’t correct while in others, the proposed structure needed a proton to be added to get the right m/z value.  What this means is that the proposed structures of the product ions aren’t the same as those actually being formed. 

So what can the lipidomics field take from this… first, the tested precursors weren’t lipids, they were amino acids and other small molecules.  Many lipids (e.g. glycerides, phospholipids, etc) follow very predictable routes to fragmentation, such as loss of ketene, carboxylate anion, headgroup, etc), and so we don’t expect to find many problems there.  However, it was interesting that some of the differences occur due to the formation of cyclic structures during fragmentation and we would expect to see such behaviour for many other lipids, such as oxylipins, or maybe also sterols.

Although the actual annotation of the fragment doesn’t form part of the spectral comparisons, it’s still important to ensure the information is correct since use of incorrect assumptions may lead to generation of additional in silico spectra that will contain significant errors.

Val O'Donnell, Cardiff University.

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25 March 2024

Lipids in Neurodiversity

As part of the Neurodiversity Celebration Week, I had a brief look into how lipids may be altered in those with an Autism Spectrum Disorder (ASD) and how lipidomics could affect a diagnosis. 

The Centers for Disease Control and Prevention US (CDC) define ASD as a “developmental disability caused by differences in the brain. People with ASD often have problems with social communication and interaction, and restricted or repetitive behaviors or interests.” There are currently no conclusive tests that can be carried out to determine ASD such as a blood or urine analysis, and diagnosis is a long and subjective process, dependent on clinical expertise. 

Various studies have suggested a contribution of altered lipid signaling and/or metabolism to the pathogenesis of ASD. A paper by Afaf El-Ansary et al. goes one step further than suggesting possible causes by depicting The Role of Lipidomics in Autism Spectrum Disorder. Here they propose that a defined set of metabolites may be useful to diagnose ASD via lipidomics. It is possible therefore that measuring lipid biomarkers may provide a novel approach to improve diagnosis for ASD.

Lauren Cockayne 

Cardiff University

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18 March 2024

New Role for Phosphoinostides: Modulation of Lysosomal Function in Response to Nutrient Status

Phosphoinositides, and their metabolism, have long been recognized as the archetype of signaling lipids.  In fact, the list of phosphoinositide-involved signaling pathways is quite impressive.  This is indeed a well-deserved reputation given the number of signaling pathways that involve these lipids.  It almost appears their roles have been exhausted but a recent report highlights yet more physiological roles for these lipids.  A recent report (Ebner et al.Cell, 2023 Nov 22; 186(24): 5328-5346) demonstrates a role for these lipids modulate activities in lysosomes that bear on the anabolic as well as catabolic functions of this organelle in response to cellular nutrient status.  The authors provide evidence that lysosome morphology and function are reversibly controlled by a nutrient-regulated signaling switch in phosphoinositide localization that modulates mTORC1.  The authors show the nutrient-dependent conversion between peripheral motile mTORC1 signaling-active and static mTORC1-inactive degradative lysosomes clustered at the cell center.  It appears that starvation triggers the relocalization of phosphatidylinositol 4-phosphate (PI(4)P)-metabolizing enzymes which reshapes the lysosomal surface proteome facilitating lysosomal proteolysis and repression of mTORC1 signaling. Additionally, lysosomal phosphatidylinositol 3-phosphate (PI(3)P), associated with motile signaling-active lysosomes in the cell periphery, is eliminated.  Important, interfering with this PI(3)P/PI(4)P lipid switch impairs the adaptive response of cells to nutrients.  These data add yet another role for phosphoinositides by showing that they play an important role in signaling a shift if cellular nutrient statues via the alteration of lysosomal membrane dynamics.

Daniel M. Raben,

The Johns Hopkins University School of Medicine, Baltimore, MD, USA

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4 March 2024

New lipids from old samples

The most widely used ionization method in LC/MS/MS, electrospray, is considered a rather “soft ionization” mode, meaning it provides limited fragmentation information for lipids, when compared with the older methods that were available on gas chromatography, such as electron impact ionization.  This has meant that detailed structural information on double bond position was not generated during fragmentation of molecules when using electrospray, and so assumptions were often made based on the most abundant fatty acyls already known to be present in the samples.  As an example, for mammalian tissues, it would be assumed that FA 20:4 was generally arachidonic acid, with its specific composition of cis double bonds at C5,8,11 and 14.   However, new MS modalities are now becoming available that, combined with LC/MS/MS can achieve fragmentation that reports on the molecular composition of lipids to a depth which was not previously possible, and these are beginning to reveal a diversity of FA in the human lipidome that goes far beyond what we thought we know.   These methods include UV-photodissociation with ozone (UVPD, or OzID), which has been implemented both on Orbitraps (Thermo) as well as ToFs, including within the ion mobility cell (Waters), and a second method called Electron activated dissociation, available on Sciex ToF instruments. The application of these approaches to lipidomics is somewhat still in its infancy and it’s going to be really interesting to see the impact of this on our understanding of the molecular diversity of lipids in general.  A recent study on this from the Blanksby group revealed unexpected diversity of lipidomes from plasma, cell lines and vernix caseosa (Nature Article).  The study increased the number of plasma FA by two-fold, including finding non-methylene interrupted FA structures, and added over 90 new FA to LMSD.  How these many new FA and the complex lipids they will undoubtedly be attached to, contribute to lipid biology and biochemistry in health and disease is going to be a matter for exciting research in the coming years.

Valerie O'Donnell, Cardiff University

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16 February 2024

Complex Sphingolipids Lipid Rafts Modulate Lipid Domains and Microlipophagy

During nutrient limitation a process known as microlipophage is induced where lipid droplets are hydrolyzed by lysosomes, or vacuoles in yeast (Saccharomyces cerevisiae).  It has been shown that cells lacking the ability to generate phase separated domains in these vacuoles are defective in macrolipophagy.  In a recent report Kim and Budin (J. Biol. Chem. 300 (1): 105496 (2024)) present some intriguing data indicating that the generation of complex sphingolipids and sorting into yeast vacuoles is key to membrane lipid phase separations in these vacuoles which modulate micro-lipophagy.  The role of these sphingolipids was examined via a systematic genetic dissection of the biosynthetic pathway of these lipids. Their data show that the abundance of complex sphingolipids determined the extent of domain formation which was associated micro-lipophagy.  Their results are the first to indicate that the trafficking of the complex sphingolipids drive membrane phase separations that impact micro-lipophagy in yeast.

Daniel M. Raben,

The Johns Hopkins University School of Medicine, Baltimore, MD, USA

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5 February 2024

The formation of a presynaptic site in a neuron requires transport of specific proteins from the soma (where the nucleus is housed) along the axon to the axon terminal.  There’s a lot not known about this process, for example, the biochemistry of the vesicles that carry the presynaptic proteins is not yet clear, nor where they originate from.  In Rizalar et al an essential role for a lipid signalling pathway in this process was recently demonstrated.   Using fluorescent labelling, chemical dimerization, and focused ion beam milling scanning electron microscopy (FIB-SEM), they showed that phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) signalling supports the transport of synaptic vesicles and their active zone proteins along the axon, guiding them to the presynaptic site located in precursor vesicles. Importantly, they also determined the ultrastructure and size of the vesicles.  PI(3,5)P2 is already known to be involved in inherited neurodegeneration, so elucidating a role in the context of axon biology extends our knowledge of how this specialised lipid is involved in brain health, thus also paving the way for improving our understanding of its role in neurological disorders.  A perspective on the article has also been published.

Valerie O'Donnell, Cardiff University

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23 January 2024

Complex Sphingolipids Lipid Rafts Modulate Lipid Domains and Microlipophagy

During nutrient limitation a process known as microlipophage is induced where lipid droplets are hydrolyzed by lysosomes, or vacuoles in yeast (Saccharomyces cerevisiae).  It has been shown that cells lacking the ability to generate phase separated domains in these vacuoles are defective in macrolipophagy.  In a recent report Kim and Budin (J. Biol. Chem. 300 (1): 105496 (2024)) present some intriguing data indicating that the generation of complex sphingolipids and sorting into yeast vacuoles is key to membrane lipid phase separations in these vacuoles which modulate micro-lipophagy.  The role of these sphingolipids was examined via a systematic genetic dissection of the biosynthetic pathway of these lipids. Their data show that the abundance of complex sphingolipids determined the extent of domain formation which was associated micro-lipophagy.  Their results are the first to indicate that the trafficking of the complex sphingolipids drive membrane phase separations that impact micro-lipophagy in yeast.

Daniel M. Raben,

The Johns Hopkins University School of Medicine, Baltimore, MD, USA

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8 January 2024

Reading about how predators use a diversity of strategies to target prey led us to a paper published last year in Science Advances by Lee et al, from Taipei, focused on how a carnivorous mushroom uses a volatile ketone lipid to paralyze and kill nematodes (https://www.science.org/doi/10.1126/sciadv.ade4809). In this interesting study, the authors used a genetic approach generating 12K random mutants of the oyster mushroom (P. ostreatus) and then tested these to identify processes for killing of C. elegans.  First, they found that the toxic mutants contained a spherical structure on their hypae, called toxocysts, and showed these were essential for paralysis of the prey.  Next, they determined the molecular composition of toxocysts using GC/MS with spectral library matching, and found that a single compound, 3-octanone, characterised these structures. Following this, in elegant studies, they then showed that 3-octanone could trigger paralysis, calcium influx and cell necrosis in the nematode, as well as disrupting cell membrane integrity and causing death.   Intriguingly, when looking at the chemical properties required for toxicity, the position of the ketone was less important than the total carbon number of the compound.  This study greatly extends our knowledge about the biology of eight-carbon volatile organic compounds in fungal biology, where they were already well known as communication signals.   Importantly, the detailed mechanisms by which these compounds act is not fully clear, but in this case, it appears that the compound inserts itself into membranes causing biophysical changes that lead to its toxic actions.  In this regard, it may be considered analogous to other toxins which disrupt membranes, such as phospholipases or bacterial pore forming toxins.  The paper proposes that this new mechanism could be useful for developing the fungus as a biocontrol agent against parasitic nematodes in agriculture.   

Valerie O'Donnell, Cardiff University.

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11 December 2023

Effect of Lipid Saturation on Nuclear Envelope Function

Since it became obvious that lipids do more than provide a platform to support membrane proteins, there has been a long-standing and growing interest among many lipid and membrane investigators regarding the role of lipids in the regulation of membrane protein function.  While most of the attention has been given to plasma membrane lipids, there has been some interest in the role of lipid residing in intracellular membranes.  Initially, much of the attention was given to the endoplasmic reticulum (ER) given its role in secretion and lipid metabolism.  In a recent article by Romanauska and Kohler (Nature Cell Biology 25:1290-1302, 2023) have provided compelling evidence that lipid acyl chain structure is linked to the structure of the double bilayer membranes that surrounds the nucleus. This double membrane is continuous with the ER.  Interestingly, their data shows that increased lipid saturation is detrimental to maintaining the homeostasis between the nuclear pore complex and the ER.  The authors argue that increases in saturated lipids leads to micron-scale lipid phase separation of the NE/ER into rigid and more elastic domains resulting in the anomalous segregation of NPCs into the elastic phase.  Importantly, the provide evidence that these phase separation makes nuclei more susceptible to rupture leading to the leakage and exposure of chromosomal DNA.  Surprisingly, lipid droplets appear to preserve the integrity of the nuclear membrane including the nuclear pore complex.  Further work on the role of lipids on the integrity and function of the nuclear envelop will certainly uncover some fundamental aspects driving the relationship between lipids and nuclear functions.

Daniel M. Raben,

The Johns Hopkins University School of Medicine, Baltimore, MD, USA

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29 November 2023

Last week, I read with interest a new paper from Anaisa Ferreira, Martin Giera and colleagues, published in Nat Comms (https://www.nature.com/articles/s41467-023-43315-x#Sec2) , showing that BCG vaccination leads to increased levels of LOX products in monocytes of healthy individuals. Importantly, this study also showed that this increase, along with changes in long-chain PUFA biosynthesis was required for the trained immunity response of these cells.  This follows on from other seminal studies showing that LOXs in monocytes and macrophages are not only involved in innate, but also adaptive immunity, however there’s still a lot we don’t know about this and what the lipids are specifically doing in this context from a signaling point of view.  Ferreira et al showed that pharmacological inhibition of FADS2, LXR, 5- or 12-LOX all reduced trained immunity in vitro, while SNPs in desaturases and LOX genes influenced trained immunity in human volunteers.  Several mono-hydroxy oxylipins were implicated in this process.  The question then becomes what are the lipids doing?  Several years ago, Stefan Uderhardt and Gerhard Krönke showed that 12/15-LOX, which is the most likely candidate for formation of many of these lipids in monocytes, was involved in immunologic tolerance (https://www.sciencedirect.com/science/article/pii/S1074761312001288?via%3Dihub). They showed how 12/15-LOX is required for removal of apoptotic cells via a mechanism involving oxidized phospholipids acting as a specific signal on the cell surface.  Although this sounds very different to the new study, both address the role of monocytic LOX in adaptive immunity and it’s tempting to speculate on how these findings maybe related. How the mono-hydroxy oxylipins detected in Ferreira et al are acting is so far unknown, and could include direct receptor dependent signaling or their esterification into complex lipid pools, as seen in Uderhardt.  Follow on work to address these questions will be very interesting.

Valerie O'Donnell, Cardiff University.

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13 November 2023

Sphingolipids are a major class of membrane lipids that play vital physiological roles, especially in the nervous system.  It has been long-established that gangliosides, the glycosphingolipids found in numerous membranes, serve as receptors for several bacterial toxins and viruses and interact with and modulate the function of other cellular receptors. The peptide (sequence CGSPGWVRC) binds to the surface of pulmonary primary vascular endothelial cells contributes to emphysema-like changes.  In a recent article by Staquicini et al. (Proc Natl Acad Sci U S A. 2023 Aug 22;120(34)) they have identified the receptor to be C16-ceramide.  These authors show that CGSPGWVRC activates acid sphingomyelinase and ceramide production, in the absence of apoptotic signaling, leading to the formation of ceramide-rich platforms.  Interestingly, these authors further showed that the targeting of the peptide to C16-ceramide can be used as a bioinorganic hydrogel for pulmonary imaging as well as a ligand-directed lung immunization tool against COVID-19. This study has provided evidence for yet another new role for sphingolipids.

Daniel M. Raben,

The Johns Hopkins University School of Medicine, Baltimore, MD, USA

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30 October 2023

Two weeks ago, via the magic of Zoom, I sat in on a lab meeting at Bruce Hammock’s lab, which was being presented by Nils Schebb, over visiting from Germany.  A large part of his talk concerned an under-researched but very interesting area…. The occurrence of oxylipins in food.  Those of us working biomedical applications of oxylipins generally think only of them as being produced endogenously. We don’t consider how we might be exposed to them through other routes, and food, especially that containing fats that have undergone some oxidation (heat, age, etc) or express lipoxygenases and other PUFA oxidizing enzymes, are an obvious potential source.  Nils focused in two recent papers, Koch et al, available here (10.1021/acs.jafc.2c04987, 10.1021/acs.jafc.3c02724) on the occurrence of C18 oxFA in flaxseed, sunflower and rapeseed oil.  He and his team identified a large number of new mono-OH lipids derived from linoleic (LA) or a-linolenic (ALA) acids, and present in higher abundance than the most often studied 9- and 13- species from either LA or ALA.  In several oils, they were present at up to 0.1% of the oil content, which is pretty significant.  We have to wonder what they might be doing there and how this might impact human health.

The work raises many new and interesting questions. Oxylipins generated enzymatically and non-enzymatically don’t only include mono-hydroxy-FA, they also include truncated reactive aldehyde species, and for PUFA with more double bonds, others with complex PG-like ring structures may also form, so what else is there? Analysis was carried out after hydrolysis to remove FA from complex lipids, so are these free oxylipins or mainly esterified to glycerides or phospholipids? Considering that refined oil consists of >99% triglycerides (fat), the oxylipins in vegetable oils should be largely bound to this species. Schebb et al also expertly used GC/MS to work out double bond position of these new lipids, so it will also be interesting to see how new LC/MS methods like UVPD might be useful in future work of this kind.

Last, what are the biological consequences once these lipids are first exposed to high acid levels in the stomach then hydrolysed to release the free oxylipins in the gut by lipases during absorption. Oxylipins have diverse biological effects, including both pro- (e.g. EP, DP, TP receptors during inflammation) and anti-inflammatory (e.g. PPARg signaling) and the likely impact of these will of course be context dependent. Much work remains to understand the implications of this work and to further our knowledge of oxylipin environmental exposure on human health.

Val O'Donnell

Cardiff University

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17 October 2023

The biophysical properties of membranes is critically important to membrane function.  This architecture can influence a number of physiologically important functions including transport, signaling, cell recognition, fusion, and interactions with the cytoskeleton.  Such biophysical properties depend on the fluidity membranes which depends on the length and degree of unsaturated fatty acid components of constituent phospholipids.  It has been recognized that the degree of saturation in eukaryotes depends on desaturases that require molecular oxygen.  This raised an interesting question as to how certain organisms, such as Schizosaccharomyces japonicus can grow in both aerobic and anaerobic conditions.  In a recent study, Panconi et al J. Biol. Chem. (2023), use microscopic, lipidomic, and molecular dynamic approaches to show that these organisms modulate membrane fluidity, at 24⁰C, but interestingly not 37⁰C, by increasing the amount of asymmetric tail phospholipids, such as 18:0 and 10:0, allowing for increased fluidity, in response to anoxic conditions.  It is noteworthy that this was not seen in a related species of yeast, Schizosaccharomyces pombe.  This gives S. japonicus a growth advantage under anoxic conditions showing once again lipids come to the rescue!

Daniel M. Raben,

The Johns Hopkins University School of Medicine, Baltimore, MD, USA

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10 October 2023

I was very interested to read Bill Christie’s recent blog on the challenges of dealing with big data in lipid research, which is nowadays exemplified by the application of informatics approaches to lipidomics.  It’s inspired me to add my own thoughts, relating to the task of assessing and ensuring data quality at the level of raw data.  The launch of new software is often accompanied by claims of its superiority in relation to existing tools.  However, it’s not till the community have time to evaluate software, that we can agree on how robust, accurate and reliable a tool really is and this can take time. Software is increasingly being asked to “adjudicate” on whether a lipid is present in a sample based on appearance of chromatographic and MS/MS data.  In the past, this was performed by visual/manual inspection which when done correctly, works very well. However, when we move to large datasets, we often need to use software to automate and increase throughput.  Great care is needed with this because computers only do as good a job as they are programmed to do, and they don’t replace common sense.  Different software tools will use distinct algorithms for the “same” job, and comparing their outputs can show wildly different results. How can we deal with this?  We have to exercise caution by sanity checking our data using our own eyes, especially where we are analysing low abundance lipids.  Understanding how a tool works “under the bonnet” is also important. Algorithms making spurious claims from raw MS data isn’t a new problem as those familiar with the story about proteomics MS in ovarian cancer biomarkers may remember.  If you haven’t heard of this, check out this study from 20 years ago, which showed that processing noise had allowed cancer patients to be distinguished from controls, using SELDI-ToF MS: https://academic.oup.com/bioinformatics/article/20/5/777/214156?login=false.  

Val O'Donnell

Cardiff University

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26 September 2023

DIESL: A New DGAT Regulated by TMX1 for the Synthesis of TAGs in Mammalian Cells

A paradigm in metabolism is the recognition that triacylglycerols (TAGs) represent a major source of stored energy in a variety of organisms from bacteria to humans.  TAGs are composed of three fatty acids esterified to the three carbons of glycerol.  There is great interest in these neutral lipids as their synthesis and metabolism play roles in a variety of physiological and pathophysiological processes.  It has been long recognized that TAGs are synthesized via in humans by the condensation of coenzyme A-conjugated fatty acids to the carbons on glycerol  This reaction has long been recognized to be catalyzed by two diacylglycerol O-acyltransferases (DGATs): DGAT1 and DGAT2.  Interestingly, other organisms possess additional enzymes for the generation of TAGs but has not been clear whether alternative pathways also exist in humans.

In a recent report by McLelland et al Nature 621:171–178 (2023) and see review by Schaffer, JC in Nature 621:47-48 (2023) has solved this mystery by identifying a novel pathway for the synthesis of TAGs in mammalian cells.  The authors performed a loss of function CRISPR screen using haploid human cells in which both DGAT1 and DGAT2 had been knocked out. In this screen, they discovered that the elimination of a transmembrane thioredoxin (TMX1) led to an increase in TAG synthesis.  As TMX1 does not catalyze the synthesis of TAGs the authors screened for genes that led to a decrease in TAG synthesis when disrupted in the haploid cells also lacking TMX1. They identified a protein they called DAG1/2-independent enzyme synthesizing storage lipids (DIESL).  Further analyses identified DIESL as another DGAT but one that is inhibited by TMX1.  Interestingly, DIESL appears to use phospholipids, or phospholipid precursors, as a source of TAG fatty acids.  This is an exciting discovery and future work to uncover the regulation and roles of this enzyme is sure to lead to other important discoveries.

Daniel M. Raben,

The Johns Hopkins University School of Medicine, Baltimore, MD, USA

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11 September 2023

As I have mentioned on several occasions, I feel that I have missed out in never having had access to modern mass spectrometry methods for analysis of intact lipids. However, I am sure that I am not alone in finding the vast amount of data that is now produced completely indigestible. I know that moves are underway to improve comparisons of data between labs, but as an independent observer I find that I can use very little of what I read in my web pages other than general conclusions. For example, in comparing lipid compositions from different species, organelles, etc, I have tabulated data from publications that are more than 50 years old, simply because I can find little comparable that is more recent.

I have just been reading an excellent review on lipidomics information, but in 27 pages and 260 references, there are no tables of compositional data or even graphical illustrations (Sarmento, M.J. et al. The expanding organelle lipidomes: current knowledge and challenges. Cell. Mol. Life Sci., 80, 237 (2023); DOI). This is not a criticism of the authors, as I understand the problem – there are simply far too many data points from each lipid in every study, especially when positional distributions in glycerolipids are taken into account. I would like to see a table in each publication (or in the supplemental information) in which the data are simplified by aggregating molecular species to give the total amount for each lipid class. Then, within each lipid class, molecular species should be tabulate as the total, saturated, monoenes, dienes, etc. Finally, the fatty acid positional distributions (and/or totals) within each glycerolipid class should be determined by aggregating the results for each molecular species. It should not be a major task to devise a computer programme to do this.

I am not advocating that the individual data points are ignored, and data must continue to be expressed as at present. My suggestion is for an additional way to present the information – not an alternative. My concern is for the external observer, who wants the big picture as well as the minutiae.

Bill Christie

The LipidWeb, Dundee, Scotland

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5 September 2023

Last week, I attended the annual meeting of the International Lipidomics Society (ILS) in Vienna. A key part of harmonization and standardization within lipidomics is the correct lipid identification and quantification by mass spectrometry. To support this, ILS is now developing minimal lipid analysis guidelines, which will describe how to properly identify and quantify lipids from raw mass spectrometry signals.  The focus will be on low abundant lipids such as oxylipins, that includes octadecanoids, eicosanoids (e.g., prostaglandins, leukotrienes, thromboxanes) and docosanoids as well as oxygenation products of n3 and n6 fatty acids termed specialized pro-resolving mediators.

I’m delighted to serve as the Chair of the new Interest Group on Oxylipin Analysis Guidelines that the International Lipidomics Society have set up. I’m grateful especially to Kim Ekroos and Gerhard Liebisch for productive and positive discussions on this topic, and for those who have agreed to act as our initial core group to set out the principles under which we will operate. These are: Makoto Arita (Riken, Japan), Craig Wheelock (Karolinska Institute, Sweden), Nils Schebb (Uni Wuppertal, Germany), Hubert Vesper (Centre for Disease Control and Prevention, USA) and Miguel Gijon (Cayman Chemical, USA). Once a plan is in place, the Interest Group will be opened up and all who are interested can join us to be part of guideline development. We encourage you to get involved by joining us on zoom and developing a published guideline.

Valerie O'Donnell

Cardiff University

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18 August 2023

Interesting Chemistry Underlying the Synthesis of GDGT in Archaea

Understanding the chemistry of lipid metabolizing enzymes is often challenging, but insights can be fascinating. Take, for example, the synthesis of isoprenoid-based ether-linked membrane lipids in Archaea.  These lipids are important because they enable these organisms to withstand extreme environmental conditions. In some archaea, like Methanocaldococcus jannaschii, it has been shown that these lipids may form macrocyclic diether lipids or macrocyclic glycerol dibiphytanyl glycerol tetraethers (GDGT). Interestingly, GDGT is a membrane-spanning lipid that contains covalent bonds between the terminal carbons of the inner and outer leaflets of the membrane, providing enhanced membrane stability. While the mechanism underlying the formation of these unique lipids was obscure for many decades, work from Squire Booker’s laboratory at The Pennsylvania State University has elucidated it (Lloyd et al. Nature. 2022 Sep;609(7925):197-203 DOI). The reaction is unique in that it involves coupling two inert sp³-hybridized carbon centers. In vitro mechanistic studies indicate that C(sp³)–C(sp³) bond formation occurs on fully saturated archaeal lipid substrates and proceeds through an intermediate containing a bond between the substrate carbon and a sulfur ion of an auxiliary [Fe₄S₄] cluster to stabilize a transient carbon-centered radical. This work finalizes the biosynthetic route for GDGT formation and reveals the first instance of C(sp³)–C(sp³) coupling in nature.

Dr. Squire Booker
Evan Pugh University Professor of Chemistry and of Biochemistry and Molecular Biology
Pennsylvania State University
Eberly College of Science
Department of Chemistry

Mr. Cody Lloyd
Pennsylvania State University
Eberly College of Science
Department of Chemistry

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7 August 2023

A few weeks ago, Matt Conroy and I had the pleasure of attending the Annual EpilipidNET meeting hosted by Justine Bertrand-Michel in Toulouse, France (https://www.epilipid.net/).  This was the penultimate annual meeting of this pan-European EU COST Network, led by Maria Fedorova and Rosario Domingues, which has over the last 3 years brought together over 390 researchers from 47 countries, far beyond Europe, to facilitate a huge range of lipidomics activities.  Huge credit is due to the leadership of EpilipidNET, for driving this initiative, which has transformed the profile of lipid research in Europe in a very short time, bringing together interest groups across diverse remits including:  Plant and Algal Lipidomics, Lipidomes of Common Model Organisms, Bacterial Lipidomes and Subcellular Lipidomics.  All these areas are of direct relevance to LIPID MAPS, since provision of data on structures, reactions pathways and lipid metadata and making this information freely available to the community is a common goal of both initiatives.  

 

If you are a young (or not so young) researcher, either experienced or new to the field, be sure to check out EpilipidNETs activities.  The last annual meeting will be in Dresden in 2024, but before this, there are several other meetings and workshops including a hackathon on curation of model organisms lipidomes.  All events and activities are free of charge to attend, and bursaries are often available too.

Valerie O'Donnell,

School of Medicine, Cardiff University, UK

 

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24 July 2023

A family of proteins referred to as the ORMs/ORMDLs serve as regulatory subunits of the rate-liming enzyme in the synthesis of sphingolipids, serine palmitoyltransferase (SPT) complex.  This complex is known to be homeostatically regulated by cellular sphingolipid levels, but how cells sense these levels has been a matter of controversy.  This controversy seems to now be resolved.  In a recent article by Xie et al (Nat. Commun. 2023, Jun 13;14(1) 3475;  DOI) the authors show that purified human SPT-ORMDL complexes are directly inhibited by ceramide.  This was accomplished by solving the cryo-EM structure of the SPT-ORMDL3 complex in a ceramide-bound state, demonstrating a specific ceramide-binding site within the complex.  Furthermore, structure-guided mutational analyses demonstrated that this ceramide binding induces and locks the N-terminus of ORMDL3 into an inhibitory conformation. Interestingly, the authors note that childhood amyotrophic lateral sclerosis (ALS) variants in the SPTLC1 subunit cause impaired ceramide sensing in the SPT-ORMDL3 mutants.  This exciting work reveals the molecular basis of ceramide sensing by SPT-ORMDL as well as the functional consequences of this interaction and suggests an important role of impaired ceramide sensing in disease.

Daniel M. Raben,

The Johns Hopkins University School of Medicine, Baltimore, MD, USA

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7 June 2023

There have been numerous studies focused on the generation and metabolism of lipid droplets. Despite these studies, our understanding of how these droplets develop from the endoplasmic reticulum (ER) has been incomplete. For example, triacylglycerol and cholesterol esters are two of the most abundant neutral lipids in these structures but they are very different molecules with very different physical properties. This is highlighted by the fact that triacylglycerols melt at -4°C while cholesterol esters melt at a much lower temperature of -44°C. A recent article from Abdou Rachid Thiam’s laboratory, and in collaboration with Ilp Vattulainen and Elina Ikonen, data are presented that indicate cholesterol esters can form supercooled lipid droplets in the presence of triacylglycerols (Nat. Commun., 14, 915 (2023);  DOI). These authors demonstrate that cholesterol esters form supercooled lipid droplets above 20 mol% with respect to triacylglycerol levels, and liquid-crystalline phases when the level increases to above 90 mol% at 37°C. They further show that at physiological temperatures, seipin-mediated triacylglycerol clusters catalyze the nucleation of cholesterol esters in the ER bilayer to initiate the formation of lipid droplets. Their data are particularly interesting given, as suggested by their melting temperatures, cholesterol esters would be expected to form a crystalline phase at physiological temperatures, but in the presence of triacylglycerols these lipids are condensed into nascent lipid droplets. Their data not only provides insights into the formation of lipid droplets, it suggests how macrophages generate cholesterol ester-rich lipid droplets leading to foam cells, as well as how spatially distinct other lipid droplets can form for other physiological processes such as steroid hormone synthesis.

Daniel M. Raben,
The Johns Hopkins University School of Medicine, Baltimore, MD, USA

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24 May 2023

Each animal cell can contain up to 1000 distinct molecular species, with each lipid class containing multiple combinations of the fatty acid components. It seems likely that a high proportion of these are simply present to provide the correct blend of physical properties required for the structural function of a lipid in membranes, but it is surprising how many individual molecular forms have been recognized as having unique biological roles within tissues. One good example of this is 1-palmitoyl-2-oleoyl-phosphatidyl-sn-glycerol of lung surfactant, which attenuates inflammation by antagonizing the cognate ligand activation of the toll-like receptors (TLR2/1, TLR3, TLR4, and TLR2/6), while it disrupts the binding of virus particles to the plasma membrane receptors required for viral uptake in host cells, including influenza and SARS-CoV-2 viruses (Numata, M. et al. The anti-inflammatory and antiviral properties of anionic pulmonary surfactant phospholipids. Immun. Rev., in press (2023);  DOI).

It has been recognised for some time that the 18:0-18:1 species of phosphatidylserine has a distinctive role in membranes probably through physical interaction with sphingolipids (Skotland, T. and Sandvig, K. The role of PS 18:0/18:1 in membrane function. Nature Commun., 10, 2752 (2019);  DOI). More surprising is a recent publication demonstrating that a bacterial species from human gut, produces a phosphatidylethanolamine species with two different branched chain components (anteiso-15:0 and iso-15:0) that has remarkable specificity for immune signalling in its host via a toll-like receptor TLR2-TLR1 heterodimer; no other combination of acyl groups works (Bae, M. et al. Akkermansia muciniphila phospholipid induces homeostatic immune responses. Nature, 608, 168-173 (2022);  DOI).

Bill Christie

The LipidWeb, Dundee, Scotland

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17 May 2023

Some biochemical terms can have needlessly complex or obscure meanings, so it is always pleasing to find terms that are immediately understandable and useful, such as ‘flippases’ and ‘scramblases’ for proteins that mediate the movement of phospholipids between the leaflets of membrane bilayers. Flippases direct phosphatidylethanolamine and phosphatidylserine to the cytoplasmic leaflet (floppases work in the opposite direction), while scramblases as the name suggests randomly scramble phospholipids between leaflets across the membrane and collapse the membrane asymmetry. In particular, the latter can transfer phosphatidylserine to the outer leaflet where its exposure on the cell surface is an ‘eat-me’ signal to macrophages, another memorable term (we Scots would call them ‘couthy’). A new review on the topic is worth a read (Sakuragi, T. and Nagata, S. Regulation of phospholipid distribution in the lipid bilayer by flippases and scramblases. Nature Rev. Mol. Cell Biol., in press (2023);  DOI).

Pick up any newspaper and you will see that artificial intelligence (AI) is giving concern for any number of reasons. I understand that one such programme passed a US bar exam with flying colours, and universities world-wide are concerned with their use to cheat in essays. There are also worries that they are being used to create bogus scientific publications, and I have seen so many review articles on ferroptosis especially lately – I will say no more! There are several commercial programmes available that purport to improve the standard of written English - a worthy objective, and I am sure that they could be of legitimate value especially for those who have difficulty with the language. Are they too open to abuse?

Bill Christie,
The LipidWeb, Dundee, Scotland

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