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The accumulation of lipid peroxides is recognized as a determinant of the occurrence of ferroptosis. However, the sensors and amplifying process of lipid peroxidation linked to ferroptosis remain obscure. Here we identify PKCβII as a critical contributor of ferroptosis through independent genome-wide CRISPR–Cas9 and kinase inhibitor library screening. Our results show that PKCβII senses the initial lipid peroxides and amplifies lipid peroxidation linked to ferroptosis through phosphorylation and activation of ACSL4. Lipidomics analysis shows that activated ACSL4 catalyses polyunsaturated fatty acid-containing lipid biosynthesis and promotes the accumulation of lipid peroxidation products, leading to ferroptosis. Attenuation of the PKCβII–ACSL4 pathway effectively blocks ferroptosis in vitro and impairs ferroptosis-associated cancer immunotherapy in vivo. Our results identify PKCβII as a sensor of lipid peroxidation, and the lipid peroxidation–PKCβII–ACSL4 positive-feedback axis may provide potential targets for ferroptosis-associated disease treatment. Through CRISPR–Cas9 and kinase inhibitor screening, Zhang et al. show that PKCβII phosphorylates and activates ACSL4 to enhance polyunsaturated fatty acid-containing lipid biosynthesis, thereby promoting accumulation of lipid peroxidation and ferroptosis.

Cells and organelles are delimited by lipid bilayers in which high deformability is essential to many cell processes, including motility, endocytosis and cell division. Membrane tension is therefore a major regulator of the cell processes that remodel membranes, albeit one that is very hard to measure in vivo. Here we show that a planarizable push–pull fluorescent probe called FliptR (fluorescent lipid tension reporter) can monitor changes in membrane tension by changing its fluorescence lifetime as a function of the twist between its fluorescent groups. The fluorescence lifetime depends linearly on membrane tension within cells, enabling an easy quantification of membrane tension by fluorescence lifetime imaging microscopy. We further show, using model membranes, that this linear dependency between lifetime of the probe and membrane tension relies on a membrane-tension-dependent lipid phase separation. We also provide calibration curves that enable accurate measurement of membrane tension using fluorescence lifetime imaging microscopy.

Cancer cells rewire their metabolism and rely on endogenous antioxidants to mitigate lethal oxidative damage to lipids. However, the metabolic processes that modulate the response to lipid peroxidation are poorly defined. Using genetic screens, we compared metabolic genes essential for proliferation upon inhibition of cystine uptake or glutathione peroxidase-4 (GPX4). Interestingly, very few genes were commonly required under both conditions, suggesting that cystine limitation and GPX4 inhibition may impair proliferation via distinct mechanisms. Our screens also identify tetrahydrobiopterin (BH4) biosynthesis as an essential metabolic pathway upon GPX4 inhibition. Mechanistically, BH4 is a potent radical-trapping antioxidant that protects lipid membranes from autoxidation, alone and in synergy with vitamin E. Dihydrofolate reductase catalyzes the regeneration of BH4, and its inhibition by methotrexate synergizes with GPX4 inhibition. Altogether, our work identifies the mechanism by which BH4 acts as an endogenous antioxidant and provides a compendium of metabolic modifiers of lipid peroxidation. Genetic screens reveal a compendium of metabolic modifiers of lipid peroxidation. Tetrahydrobiopterin is essential under GPX4 inhibition, acting as a radical-trapping antioxidant that inhibits lipid peroxidation and is regenerated by DHFR.

Ferroptosis is iron-dependent oxidative cell death. Labile iron and polyunsaturated fatty acid (PUFA)-containing lipids are two critical factors for ferroptosis execution. Many processes regulating iron homeostasis and lipid synthesis are critically involved in ferroptosis. However, it remains unclear whether biological processes other than iron homeostasis and lipid synthesis are associated with ferroptosis. Using kinase inhibitor library screening, we discovered a small molecule named CGI1746 that potently blocks ferroptosis. Further studies demonstrate that CGI1746 acts through sigma-1 receptor (σ 1 R), a chaperone primarily located at mitochondria-associated membranes (MAMs), to inhibit ferroptosis. Suppression of σ 1 R protects mice from cisplatin-induced acute kidney injury hallmarked by ferroptosis. Mechanistically, CGI1746 treatment or genetic disruption of MAMs leads to defective Ca 2+ transfer, mitochondrial reactive oxygen species (ROS) production and PUFA-containing triacylglycerol accumulation. Therefore, we propose a critical role for MAMs in ferroptosis execution. A small molecule, CGI1746, was identified to block ferroptosis acting through the sigma-1 receptor, a chaperone primarily at endoplasmic reticulum–mitochondria contacts to mediate calcium transfer.

Maintenance of lipid asymmetry across the two leaflets of the plasma membrane (PM) bilayer is a ubiquitous feature of eukaryotic cells. Loss of this asymmetry has been widely associated with cell death. However, increasing evidence points to the physiological importance of non-apoptotic, transient changes in PM asymmetry. Such transient scrambling events are associated with a range of biological functions, including intercellular communication and intracellular signaling. Thus, regulation of interleaflet lipid distribution in the PM is a broadly important but underappreciated cellular process with key physiological and structural consequences. Here, we compile the mounting evidence revealing multifaceted, functional roles of PM asymmetry and transient loss thereof. We discuss the consequences of reversible asymmetry on PM structure, biophysical properties and interleaflet coupling. We argue that despite widespread recognition of broad aspects of membrane asymmetry, its importance in cell biology demands more in-depth investigation of its features, regulation, and physiological and pathological implications. The asymmetric distribution of lipids, including cholesterol, in biological membranes established actively by flippases and scramblases has structural, biophysical and functional consequences in cells and implications for communication across membranes.

Whether stem-cell-like cancer cells avert ferroptosis to mediate therapy resistance remains unclear. In this study, using a soft fibrin gel culture system, we found that tumor-repopulating cells (TRCs) with stem-cell-like cancer cell characteristics resist chemotherapy and radiotherapy by decreasing ferroptosis sensitivity. Mechanistically, through quantitative mass spectrometry and lipidomic analysis, we determined that mitochondria metabolic kinase PCK2 phosphorylates and activates ACSL4 to drive ferroptosis-associated phospholipid remodeling. TRCs downregulate the PCK2 expression to confer themselves on a structural ferroptosis-resistant state. Notably, in addition to confirming the role of PCK2-pACSL4(T679) in multiple preclinical models, we discovered that higher PCK2 and pACSL4(T679) levels are correlated with better response to chemotherapy and radiotherapy as well as lower distant metastasis in nasopharyngeal carcinoma cohorts. Phosphorylation of ACSL4 by mitochondria-located metabolic kinase PCK2 is critical to regulating ferroptosis-associated phospholipid remodeling in tumor-repopulating cells that are resistant to chemotherapy and radiotherapy.

Monolyso-cardiolipin (MLCL) accumulates in individuals with Barth syndrome, but this can be mitigated by stabilization of cardiolipin from metabolism to MLCL via assembly into supercomplexes of oxidative phosphorylation proteins. This process was found to be defective in subjects with Barth syndrome. Cardiolipin is a specific mitochondrial phospholipid that has a high affinity for proteins and that stabilizes the assembly of supercomplexes involved in oxidative phosphorylation. We found that sequestration of cardiolipin in protein complexes is critical to protect it from degradation. The turnover of cardiolipin is slower by almost an order of magnitude than the turnover of other phospholipids. However, in subjects with Barth syndrome, cardiolipin is rapidly degraded via the intermediate monolyso-cardiolipin. Treatments that induce supercomplex assembly decrease the turnover of cardiolipin and the concentration of monolyso-cardiolipin, whereas dissociation of supercomplexes has the opposite effect. Our data suggest that cardiolipin is uniquely protected from normal lipid turnover by its association with proteins, but this association is compromised in subjects with Barth syndrome, leading cardiolipin to become unstable, which in turn causes the accumulation of monolyso-cardiolipin.

Lipid droplets (LDs) feature a unique monolayer lipid membrane that has not been extensively studied due to the lack of suitable molecular probes that are able to distinguish this membrane from the LD lipid core. In this work, we present a three-pronged molecular probe design strategy that combines lipophilicity-based organelle targeting with microenvironment-dependent activation and design an LD membrane labeling pro-probe called LDM . Upon activation by the HClO/ClO − microenvironment that surrounds LDs, LDM pro-probe releases LDM-OH probe that binds to LD membrane proteins thus enabling visualization of the ring-like LD membrane. By utilizing LDM , we identify the dynamic mechanism of LD membrane contacts and their protein accumulation parameters. Taken together, LDM represents the first molecular probe for imaging LD membranes in live cells to the best of our knowledge, and represents an attractive tool for further investigations into the specific regulatory mechanisms with LD-related metabolism diseases and drug screening. Lipid droplets (LDs) and their membrane proteins play essential roles in many biological processes, but the monolayer lipid membrane of LDs has not been extensively studied due to the lack of suitable molecular probes. Here, the authors report an LD membrane labelling pro-probe called LDM, which, upon activation by the HClO/ClO− microenvironment that surrounds LDs, undergoes a color change and releases LDM-OH probe that binds to LD membrane proteins.

Intracellular recognition of lipopolysaccharide (LPS) by mouse caspase-11 or human caspase-4 is a vital event for the activation of the noncanonical inflammasome. Whether negative regulators are involved in intracellular LPS sensing is still elusive. Here we show that adipose triglyceride lipase (ATGL) is a negative regulator of the noncanonical inflammasome. Through screening for genes participating in the noncanonical inflammasome, ATGL is identified as a negative player for intracellular LPS signaling. ATGL binds LPS and catalyzes the removal of the acylated side chains that contain ester bonds. LPS with under-acylated side chains no longer activates the inflammatory caspases. Cells with ATGL deficiency exhibit enhanced immune responses when encountering intracellular LPS, including an elevated secretion of interleukin-1β, decreased cell viability and increased cell cytotoxicity. Moreover, ATGL-deficient mice show exacerbated responses to endotoxin challenges. Our results uncover that ATGL degrades cytosolic LPS to suppress noncanonical inflammasome activation. Adipose triglyceride lipase (ATGL), an enzyme in fatty acid metabolism, was identified as a negative regulator of the noncanonical inflammasome. ATGL binds to lipopolysaccharide and catalyzes the hydrolysis of fatty acid side chains blocking inflammasome activation.

Non-vesicular lipid transport between bilayers at membrane contact sites plays important physiological roles. Mechanistic insight into the action of lipid-transport proteins localized at these sites requires determination of the distance between bilayers at which this transport can occur. Here we developed DNA-origami nanostructures to organize size-defined liposomes at precise distances and used them to study lipid transfer by the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain of extended synaptotagmin 1 (E-Syt1). Pairs of DNA-ring-templated donor and acceptor liposomes were docked through DNA pillars, which determined their distance. The SMP domain was anchored to donor liposomes via an unstructured linker, and lipid transfer was assessed via a Förster resonance energy transfer (FRET)-based assay. We show that lipid transfer can occur over distances that exceed the length of an SMP dimer, which is compatible with the shuttle model of lipid transport. The DNA nanostructures developed here can also be adapted to study other processes occurring where two membranes are closely apposed to each other. Use of DNA-origami nanostructures to study lipid transfer between closely apposed membrane bilayers supports a model where phospholipids are transferred by extended synaptotagmin 1 between the endoplasmic reticulum and plasma membrane through a shuttle mechanism.