Cardiolipin (CL) is a four acyl chain, mitochondrial-specific phospholipid crucial for maintenance of inner mitochondrial membrane (IMM) structure and function. In healthy tissues, CL acyl chains are highly unsaturated and maintained by a conserved remodeling pathway. However, dysregulation of CL acyl chain composition can arise from mutations in the CL transacylase, Tafazzin (TAZ), resulting in Barth syndrome (BTHS), where patients exhibit heightened mitochondrial dysfunction. Cells lacking TAZ accumulate three acyl chain monolysocardiolipin (MLCL) as well as CL species with saturated acyl chains (CLsat). While the presence of MLCL destabilizes electron transport chain (ETC) complexes and IMM-shaping proteins, the contributions of CLsat to mitochondrial dysfunction have not been elucidated. Here, we find that treatment of TAZ knockout cells with exogenous saturated fatty acids causes accumulation of CLsat and loss of IMM structure despite only minimal changes in MLCL composition. Cells with elevated CLsat show reduced fluidity of the inner membrane measured by a solvatochromic probe. Biophysical measurements and molecular dynamics analyses showed that di-saturated (C16:0 18:1)2 CL species order and rigidify membranes, while also losing the intrinsic lipid curvature characteristic of tetra-unsaturated CL. These results implicate CLsat as a potential driver of mitochondrial dysfunction and an additional therapeutic target in mitigating BTHS pathology.

As lipidomics approaches its 25th anniversary, we explore how lipid research has matured over the years while highlighting emerging innovations that are expanding our ability to study these diverse, life-critical biomolecules. In particular, we showcase the community-driven, open-access databases, software, and educational resources made freely available through the ELIXIR Core Data Resource LIPID MAPS for the benefit of both established and new researchers.

Membranes contain thousands of different lipids, but it is poorly understood why their compositions vary across cell types and environments. We report that lipid metabolism balances the molecular curvature of phospholipids, a parameter that describes their shape and propensity to destabilize flat sheets. We utilized extreme hydrostatic pressures—similar to those found in the deep ocean—to change the shape (reduce the curvature) of phospholipids in growing cells. Yeast and human cells respond to this stress by increasing synthesis of distinct high-curvature lipid species. The results support a model in which eukaryotic cells actively regulate lipid composition to maintain their membranes in a frustrated state, a dynamic that could be important for maintaining core functions in membrane trafficking.

mTORi induces ferritinophagy in HNSCC cells. (Related to Figure 3)

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain of glycerophospholipids. Polyunsaturated fatty acids (PUFAs) are preferentially attached at the sn-2 position of glycerophospholipids and are easily truncated by oxidation. The truncated-oxidized phospholipids (tr-oxPLs) trigger various cellular responses, and PLA2s may play a critical role in the metabolism of the tr-oxPLs by removing the oxidized sn-2 chain. In the present study, we demonstrated using an in vitro lipidomics assay that Group VIA calcium-independent PLA2 (GVIA iPLA2) showed high activity toward phosphatidylcholine with a 9-oxononanoyl chain, but not with an azelaoyl chain on the sn-2 position. We conducted molecular dynamics simulations which revealed that the hydrophilicity of the sn-2 acyl chain critically affects the binding of the substrate in the active site. Based on the unique specificity of GVIA iPLA2 toward tr-oxPLs, we synthesized an oxidatively modified inhibitor (GK766) for GVIA iPLA2, aiming for improvement of its selectivity and/or potency. As we expected, the modified inhibitor improved its selectivity of GVIA iPLA2 compared to the unmodified inhibitor (GK187), although the inhibitory effect became somewhat weaker. More importantly, we demonstrated that GK766 induces cell death by ferroptosis more effectively than GK187 using an erythroleukemia cell line. In the present study, we have further defined the unique substrate specificity of GVIA iPLA2 toward tr-oxPLs and its molecular mechanism. Furthermore, we have developed a novel specificity-based inhibitor that induces ferroptosis demonstrating that using substrate selectivity helps in developing more effective therapeutics.

Sensitazing hits selected from CRISPR screening

Insulin resistance accompanied by hepatic steatosis is a common complication of obesity. In an effort to identify plasma lipids that could be biomarkers or causes of insulin resistance with steatosis in people with obesity, we evaluated the plasma lipidome in three distinct groups separated by adiposity, hepatic steatosis, and insulin sensitivity, assessed by using the hyperinsulinemic-euglycemic clamp procedure: i) insulin-sensitive lean (ISL, n = 13); ii) insulin-sensitive obese (ISO, n = 14); and iii) insulin-resistant obese with hepatic steatosis (IROS, n = 13). We evaluated 759 complex lipid species in 16 subclasses (including phospholipids, glycerolipids, sphingolipids, acylcarnitines, and cholesteryl esters) and 84 eicosanoids in fasting plasma samples. Total abundances of each lipid subclass (sum of species) in the ISO group were not different from values in the ISL group, whereas phosphatidylethanolamines, triglycerides, and diacylglycerols were more abundant in the IROS than in the ISO group. The abundances of only 5 individual complex lipid species were different between the ISL and ISO groups, whereas the abundances of 23 lipids were different between the ISO and IROS groups. More complex lipids were associated with insulin sensitivity (n = 124) than obesity per se (n = 7). In contrast, plasma eicosanoids were not different between the ISO and IROS groups but were greater in both groups with obesity than in the ISL group. We conclude that insulin resistance with hepatic steatosis is associated with alterations in the plasma complex lipidome, independent of adiposity, in people with obesity, whereas adiposity has a greater impact than insulin resistance on plasma eicosanoid concentrations.

Cell membranes are composed of both bilayer-supporting and non-bilayer phospholipids, with the latter's negative intrinsic curvature aiding in membrane trafficking and the dynamics of membrane proteins. Phospholipid metabolism has long been recognized to maintain membrane fluidity, but whether it also acts to maintain the function of high-curvature lipids is not resolved. Here, we find that cells grown under hydrostatic pressure - used to artificially reduce lipid curvature - maintain lipidome curvature through metabolic acclimation. We first observed that manipulation of the lipidome curvature via the phosphatidylethanolamine (PE) to phosphatidylcholine (PC) ratio affects high-pressure growth and viability of yeast independently of membrane fluidity. In wild-type cells, X-ray scattering measurements revealed an increased propensity for lipid extracts to form non-lamellar phases after extended pressure incubations. Unexpectedly, this change in phase behavior was not due to increased levels of PE, but of phosphatidylinositol (PI), the only major phospholipid class whose curvature had not been previously characterized. We found that PI is a non-bilayer lipid, with a negative curvature intermediate to that of PE and PC. Accounting for PI, mean lipidome curvature was defended in response to pressure by two distantly related yeasts. Lipidome curvature also responded to pressure in a human cancer cell line through ether phospholipid metabolism and chain remodeling, but not in bacterial cells. These findings indicate that eukaryotic phospholipid metabolism uses diverse mechanisms to maintain curvature frustration in cell membranes.

Eicosanoids are key players in inflammatory diseases and cancer. Targeting their production by inhibiting Group IVA cytosolic phospholipase A2 (cPLA2α) offers a promising approach for cancer therapy. In this study, we synthesize a second generation of thiazolyl ketone inhibitors of cPLA2α starting with compound GK470 (AVX235) and test their in vitro and cellular activities. We identify a more potent and selective lead molecule, GK420 (AVX420), which we test in parallel with AVX235 and a structurally unrelated compound, AVX002 for inhibition of cell viability across a panel of cancer cell lines. From this, we show that activity of polycomb group repressive complex 2 is a key molecular determinant of sensitivity to cPLA2α inhibition, while resistance depends on antioxidant response pathways. Consistent with these results, we show that elevated intracellular reactive oxygen species and activating transcription factor 4 target gene expression precede cell death in AVX420-sensitive T-cell acute lymphoblastic leukemia cells. Our findings imply cPLA2α may support cancer by mitigating oxidative stress and inhibiting tumor suppressor expression and suggest that AVX420 has potential for treating acute leukemias and other cancers that are susceptible to oxidative cell death.

Cytosolic phospholipase A2 (cPLA2) associates with membranes, where it hydrolyzes phospholipids containing arachidonic acid to initiate an inflammatory cascade. All-atom molecular dynamics simulations were employed to understand the activation process when cPLA2 associates with the endoplasmic reticulum (ER) membrane of macrophages, where it acts. We found that membrane association causes the lid region of cPLA2 to undergo a closed-to-open state transition that is accompanied by the sideways movement of loop 495-540, allowing the exposure of a cluster of lysine residues (K488, K541, K543, and K544), which are known to bind allosteric activator PIP2 from the membrane. The active site of the open form of cPLA2, containing catalytic dyad residues S228 and D549, exhibited a 3-fold larger cavity than the closed form of cPLA2 in aqueous solution. These findings provide mechanistic insight into how cPLA2-ER membrane association promotes major transitions between conformational states critical to allosteric activation and enzymatic phospholipid hydrolysis.

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

Identifying carbon-carbon double bond (C=C) positions in complex lipids is essential for elucidating physiological and pathological processes. Currently, this is impossible in high-throughput analyses of native lipids without specialized instrumentation that compromises ion yields. Here, we demonstrate automated, chain-specific identification of C=C positions in complex lipids based on the retention time derived from routine reverse-phase chromatography tandem mass spectrometry (RPLC-MS/MS). We introduce LC=CL, a computational solution that utilizes a comprehensive database capturing the elution profile of more than 2400 complex lipid species identified in RAW264.7 macrophages, including 1145 newly reported compounds. Using machine learning, LC=CL provides precise and automated C=C position assignments, adaptable to any suitable chromatographic condition. To illustrate the power of LC=CL, we re-evaluated previously published data and discovered new C=C position-dependent specificity of cytosolic phospholipase A2 (cPLA2). Accordingly, C=C position information is now readily accessible for large-scale high-throughput studies with any MS/MS instrumentation and ion activation method.

Genomic alterations converging on persistent activation of the PI3K/mTOR pathway represent one of the most frequently altered signaling circuitries in cancer. However, the clinical efficacy of mTOR inhibitors (mTORi) has been limited. In this study, we took advantage of the widespread activation of PI3K/mTOR signaling in head and neck squamous cell carcinoma (HNSCC) and the promising effects of mTORi in HNSCC experimental models and recent clinical trials to gain a mechanistic understanding of the antitumoral activity of mTORi. A genome-wide CRISPR screen revealed that treatment with mTORi promotes the autophagic degradation of ferritin (ferritinophagy), consequently increasing free intracellular iron, inducing lipid peroxidation, and ultimately driving cancer cell demise by ferroptosis. These findings provide a rationale for synergistic combinations repurposing approved drugs that disable cellular ferroptotic defense mechanisms. Together, this study provides a molecular framework underlying the antitumor activity of mTORi in HNSCC, thereby revealing multimodal precision therapies for HNSCC and many human malignancies displaying overactive PI3K/mTOR signaling.Significance: Inhibition of mTOR induces ferritinophagy that increases free iron and stimulates ferroptosis, suggesting that this axis could be harnessed to help predict responses and to develop rational combination therapies to overcome resistance.

Free fatty acid analysis comparing control and INK128 treatment

Biomedical research on the brain has led to many discoveries and developments, such as understanding human consciousness and the mind and overcoming brain diseases. However, historical biomedical research on the brain has unique characteristics that differ from those of conventional biomedical research. For example, there are different scientific interpretations due to the high complexity of the brain and insufficient intercommunication between researchers of different disciplines owing to the limited conceptual and technical overlap of distinct backgrounds. Therefore, the development of biomedical research on the brain has been slower than that in other areas. Brain biomedical research has recently undergone a paradigm shift, and conducting patient-centered, large-scale brain biomedical research has become possible using emerging high-throughput analysis tools. Neuroimaging, multiomics, and artificial intelligence technology are the main drivers of this new approach, foreshadowing dramatic advances in translational research. In addition, emerging interdisciplinary cooperative studies provide insights into how unresolved questions in biomedicine can be addressed. This review presents the in-depth aspects of conventional biomedical research and discusses the future of biomedical research on the brain.

The conformational molecular dynamics of cytosolic phospholipase A2 (cPLA2) when interacting and associating with a simple phospholipid bilayer (Mouchlis et al. PNAS 2015) and macrophage membranes representing an average phospholipid composition (Mouchlis et al. JACS 2018) have been previously reported. We have now carried out molecular dynamics studies focused on the endoplasmic reticulum membrane in RAW macrophages where cPLA2 resides when carrying out enzymatic hydrolysis of its membrane phospholipids. Our current work also employed classical all-atom molecular dynamics simulations, but on much larger and more heterogeneous membranes. An extensive UPLC/MS study of the lipidome of RAW macrophage's in different subcellular organelles (Andreyev, et al. JLR 2010) had inspired us to build a more relevant membrane system and larger than previously accomplished by using more than 600 K atoms and an explicit solvent model. The interaction of the C2 domain and the catalytic domain were differentiated by simulating cPLA2 in both an aqueous solution and in a membrane-bound state. Our work also examined the role of phosphatidylinositol 4,5-bisphosphate (PIP2) in the membranes during interaction of cPLA2 with the ER membrane. These findings are important in establishing how cPLA2 functions in a more relevant membrane system and our understanding of membrane-induced allostery in PLA2's (Mouchlis and Dennis, Accounts Chem Res 2022).

Pedigree analysis, clinical, gross, microscopic, ultrastructural, and lipidomic findings in 4 female superb bird-of-paradise (SBOP, Lophorina superba) siblings led to the diagnosis of a primary inherited glycerolipid storage disease. These birds were the offspring of a related breeding pair (inbreeding coefficient = 0.1797) and are the only known SBOPs to display this constellation of lesions. The birds ranged from 0.75 to 4.3 years of age at the time of death. Two birds were euthanized and 1 died naturally due to the disease, and 1 died of head trauma with no prior clinical signs. Macroscopic findings included hepatomegaly and pallor (4/4), cardiac and renal pallor (2/4), and coelomic effusion (1/4). Microscopic examination found marked tissue distortion due to cytoplasmic lipid vacuoles in hepatocytes (4/4), cardiomyocytes (4/4), renal tubular epithelial cells (4/4), parathyroid gland principal cells (2/2), exocrine pancreatic cells (3/3), and the glandular cells of the ventriculus and proventriculus (3/3). Ultrastructurally, the lipids were deposited in single to coalescing or fused droplets lined by an inconspicuous or discontinuous monolayer membrane. Lipidomic profiling found that the cytoplasmic lipid deposits were primarily composed of triacylglycerols. Future work, including sequencing of the SBOP genome and genotyping, will be required to definitively determine the underlying genetic mechanism of this disease.