The germline coordinates mitokine signaling.

The ability of mitochondria to coordinate stress responses across tissues is critical for health. In C. elegans, neurons experiencing mitochondrial stress elicit an inter-tissue signaling pathway through the release of mitokine signals, such as serotonin or the Wnt ligand EGL-20, which activate the mitochondrial unfolded protein response (UPRMT) in the periphery to promote organismal health and lifespan. We find that germline mitochondria play a surprising role in neuron-to-periphery UPRMT signaling. Specifically, we find that germline mitochondria signal downstream of neuronal mitokines, Wnt and serotonin, and upstream of lipid metabolic pathways in the periphery to regulate UPRMT activation. We also find that the germline tissue itself is essential for UPRMT signaling. We propose that the germline has a central signaling role in coordinating mitochondrial stress responses across tissues, and germline mitochondria play a defining role in this coordination because of their inherent roles in germline integrity and inter-tissue signaling.

Phospholipids with two polyunsaturated fatty acyl tails promote ferroptosis.

Phospholipids containing a single polyunsaturated fatty acyl tail (PL-PUFA1s) are considered the driving force behind ferroptosis, whereas phospholipids with diacyl-PUFA tails (PL-PUFA2s) have been rarely characterized. Dietary lipids modulate ferroptosis, but the mechanisms governing lipid metabolism and ferroptosis sensitivity are not well understood. Our research revealed a significant accumulation of diacyl-PUFA phosphatidylcholines (PC-PUFA2s) following fatty acid or phospholipid treatments, correlating with cancer cell sensitivity to ferroptosis. Depletion of PC-PUFA2s occurred in aging and Huntington's disease brain tissue, linking it to ferroptosis. Notably, PC-PUFA2s interacted with the mitochondrial electron transport chain, generating reactive oxygen species (ROS) for initiating lipid peroxidation. Mitochondria-targeted antioxidants protected cells from PC-PUFA2-induced mitochondrial ROS (mtROS), lipid peroxidation, and cell death. These findings reveal a critical role for PC-PUFA2s in controlling mitochondria homeostasis and ferroptosis in various contexts and explain the ferroptosis-modulating mechanisms of free fatty acids. PC-PUFA2s may serve as diagnostic and therapeutic targets for modulating ferroptosis.

Large neutral amino acid levels tune perinatal neuronal excitability and survival.

Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.

Complex effects of sequence variants on lipid levels and coronary artery disease.

Many sequence variants have additive effects on blood lipid levels and, through that, on the risk of coronary artery disease (CAD). We show that variants also have non-additive effects and interact to affect lipid levels as well as affecting variance and correlations. Variance and correlation effects are often signatures of epistasis or gene-environmental interactions. These complex effects can translate into CAD risk. For example, Trp154Ter in FUT2 protects against CAD among subjects with the A1 blood group, whereas it associates with greater risk of CAD in others. His48Arg in ADH1B interacts with alcohol consumption to affect lipid levels and CAD. The effect of variants in TM6SF2 on blood lipids is greatest among those who never eat oily fish but absent from those who often do. This work demonstrates that variants that affect variance of quantitative traits can allow for the discovery of epistasis and interactions of variants with the environment.

Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

Lipid Transport Across Bacterial Membranes.

The movement of lipids within and between membranes in bacteria is essential for building and maintaining the bacterial cell envelope. Moving lipids to their final destination is often energetically unfavorable and does not readily occur spontaneously. Bacteria have evolved several protein-mediated transport systems that bind specific lipid substrates and catalyze the transport of lipids across membranes and from one membrane to another. Specific protein flippases act in translocating lipids across the plasma membrane, overcoming the obstacle of moving relatively large and chemically diverse lipids between leaflets of the bilayer. Active transporters found in double-membraned bacteria have evolved sophisticated mechanisms to traffic lipids between the two membranes, including assembling to form large, multiprotein complexes that resemble bridges, shuttles, and tunnels, shielding lipids from the hydrophilic environment of the periplasm during transport. In this review, we explore our current understanding of the mechanisms thought to drive bacterial lipid transport.

Comprehensive Multi-omics Analysis Reveals Mitochondrial Stress as a Central Biological Hub for Spaceflight Impact.

Spaceflight is known to impose changes on human physiology with unknown molecular etiologies. To reveal these causes, we used a multi-omics, systems biology analytical approach using biomedical profiles from fifty-nine astronauts and data from NASA's GeneLab derived from hundreds of samples flown in space to determine transcriptomic, proteomic, metabolomic, and epigenetic responses to spaceflight. Overall pathway analyses on the multi-omics datasets showed significant enrichment for mitochondrial processes, as well as innate immunity, chronic inflammation, cell cycle, circadian rhythm, and olfactory functions. Importantly, NASA's Twin Study provided a platform to confirm several of our principal findings. Evidence of altered mitochondrial function and DNA damage was also found in the urine and blood metabolic data compiled from the astronaut cohort and NASA Twin Study data, indicating mitochondrial stress as a consistent phenotype of spaceflight.

A Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in Tomato.

In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.

Membrane Protein-Lipid Interactions Probed Using Mass Spectrometry.

Membrane proteins that exist in lipid bilayers are not isolated molecular entities. The lipid molecules that surround them play crucial roles in maintaining their full structural and functional integrity. Research directed at investigating these critical lipid-protein interactions is developing rapidly. Advancements in both instrumentation and software, as well as in key biophysical and biochemical techniques, are accelerating the field. In this review, we provide a brief outline of structural techniques used to probe protein-lipid interactions and focus on the molecular aspects of these interactions obtained from native mass spectrometry (native MS). We highlight examples in which lipids have been shown to modulate membrane protein structure and show how native MS has emerged as a complementary technique to X-ray crystallography and cryo-electron microscopy. We conclude with a short perspective on future developments that aim to better understand protein-lipid interactions in the native environment.

Lysosomal Glycosphingolipid Storage Diseases.

Glycosphingolipids are cell-type-specific components of the outer leaflet of mammalian plasma membranes. Gangliosides, sialic acid-containing glycosphingolipids, are especially enriched on neuronal surfaces. As amphi-philic molecules, they comprise a hydrophilic oligosaccharide chain attached to a hydrophobic membrane anchor, ceramide. Whereas glycosphingolipid formation is catalyzed by membrane-bound enzymes along the secretory pathway, degradation takes place at the surface of intralysosomal vesicles of late endosomes and lysosomes catalyzed in a stepwise fashion by soluble hydrolases and assisted by small lipid-binding glycoproteins. Inherited defects of lysosomal hydrolases or lipid-binding proteins cause the accumulation of undegradable material in lysosomal storage diseases (GM1 and GM2 gangliosidosis; Fabry, Gaucher, and Krabbe diseases; and metachromatic leukodystrophy). The catabolic processes are strongly modified by the lipid composition of the substrate-carrying membranes, and the pathological accumulation of primary storage compounds can trigger an accumulation of secondary storage compounds (e.g., small glycosphingolipids and cholesterol in Niemann-Pick disease).

Lipid Cell Biology: A Focus on Lipids in Cell Division.

Cells depend on hugely diverse lipidomes for many functions. The actions and structural integrity of the plasma membrane and most organelles also critically depend on membranes and their lipid components. Despite the biological importance of lipids, our understanding of lipid engagement, especially the roles of lipid hydrophobic alkyl side chains, in key cellular processes is still developing. Emerging research has begun to dissect the importance of lipids in intricate events such as cell division. This review discusses how these structurally diverse biomolecules are spatially and temporally regulated during cell division, with a focus on cytokinesis. We analyze how lipids facilitate changes in cellular morphology during division and how they participate in key signaling events. We identify which cytokinesis proteins are associated with membranes, suggesting lipid interactions. More broadly, we highlight key unaddressed questions in lipid cell biology and techniques, including mass spectrometry, advanced imaging, and chemical biology, which will help us gain insights into the functional roles of lipids.

Ancient Biomolecules and Evolutionary Inference.

Over the past three decades, studies of ancient biomolecules-particularly ancient DNA, proteins, and lipids-have revolutionized our understanding of evolutionary history. Though initially fraught with many challenges, today the field stands on firm foundations. Researchers now successfully retrieve nucleotide and amino acid sequences, as well as lipid signatures, from progressively older samples, originating from geographic areas and depositional environments that, until recently, were regarded as hostile to long-term preservation of biomolecules. Sampling frequencies and the spatial and temporal scope of studies have also increased markedly, and with them the size and quality of the data sets generated. This progress has been made possible by continuous technical innovations in analytical methods, enhanced criteria for the selection of ancient samples, integrated experimental methods, and advanced computational approaches. Here, we discuss the history and current state of ancient biomolecule research, its applications to evolutionary inference, and future directions for this young and exciting field.

Architectures of Lipid Transport Systems for the Bacterial Outer Membrane.

How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.

Lipid Droplet Biogenesis.

Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids for energy or membrane synthesis and act as hubs for metabolic processes. Cells generate LDs de novo, converting cells to emulsions with LDs constituting the dispersed oil phase in the aqueous cytoplasm. Here we review our current view of LD biogenesis. We present a model of LD formation from the ER in distinct steps and highlight the biology of proteins that govern this biophysical process. Areas of incomplete knowledge are identified, as are connections with physiology and diseases linked to alterations in LD biology.

Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis.

The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.

Uptake of oxidized lipids by the scavenger receptor CD36 promotes lipid peroxidation and dysfunction in CD8+ T cells in tumors.

A common metabolic alteration in the tumor microenvironment (TME) is lipid accumulation, a feature associated with immune dysfunction. Here, we examined how CD8+ tumor infiltrating lymphocytes (TILs) respond to lipids within the TME. We found elevated concentrations of several classes of lipids in the TME and accumulation of these in CD8+ TILs. Lipid accumulation was associated with increased expression of CD36, a scavenger receptor for oxidized lipids, on CD8+ TILs, which also correlated with progressive T cell dysfunction. Cd36-/- T cells retained effector functions in the TME, as compared to WT counterparts. Mechanistically, CD36 promoted uptake of oxidized low-density lipoproteins (OxLDL) into T cells, and this induced lipid peroxidation and downstream activation of p38 kinase. Inhibition of p38 restored effector T cell functions in vitro, and resolution of lipid peroxidation by overexpression of glutathione peroxidase 4 restored functionalities in CD8+ TILs in vivo. Thus, an oxidized lipid-CD36 axis promotes intratumoral CD8+ T cell dysfunction and serves as a therapeutic avenue for immunotherapies.

Lipid metabolism in sickness and in health: Emerging regulators of lipotoxicity.

Lipids play crucial roles in signal transduction, contribute to the structural integrity of cellular membranes, and regulate energy metabolism. Questions remain as to which lipid species maintain metabolic homeostasis and which disrupt essential cellular functions, leading to metabolic disorders. Here, we discuss recent advances in understanding lipid metabolism with a focus on catabolism, synthesis, and signaling. Technical advances, including functional genomics, metabolomics, lipidomics, lipid-protein interaction maps, and advances in mass spectrometry, have uncovered new ways to prioritize molecular mechanisms mediating lipid function. By reviewing what is known about the distinct effects of specific lipid species in physiological pathways, we provide a framework for understanding newly identified targets regulating lipid homeostasis with implications for ameliorating metabolic diseases.

Exploring membrane asymmetry and its effects on membrane proteins.

Plasma membranes utilize free energy to maintain highly asymmetric, non-equilibrium distributions of lipids and proteins between their two leaflets. In this review we discuss recent progress in quantitative research enabled by using compositionally controlled asymmetric model membranes. Both experimental and computational studies have shed light on the nuanced mechanisms that govern the structural and dynamic coupling between compositionally distinct bilayer leaflets. This coupling can increase the membrane bending rigidity and induce order - or lipid domains - across the membrane. Furthermore, emerging evidence indicates that integral membrane proteins not only respond to asymmetric lipid distributions but also exhibit intriguing asymmetric properties themselves. We propose strategies to advance experimental research, aiming for a deeper, quantitative understanding of membrane asymmetry, which carries profound implications for cellular physiology.

The DDHD2-STXBP1 interaction mediates long-term memory via generation of saturated free fatty acids.

The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brain's lipid landscape remain largely unexplored. The levels of saturated FFAs, particularly of myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the PLA1 isoform DDHD2 in mice dramatically reduces saturated FFA responses to memory acquisition across the brain. Furthermore, DDHD2 loss also decreases memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits that mirror human spastic paraplegia. Via pulldown-mass spectrometry analyses, we find that DDHD2 binds to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficient STXBP1+/- mouse model of human early infantile encephalopathy associated with intellectual disability and motor dysfunction, we show that STXBP1 controls targeting of DDHD2 to the plasma membrane and generation of saturated FFAs in the brain. These findings suggest key roles for DDHD2 and STXBP1 in lipid metabolism and in the processes of synaptic plasticity, learning, and memory.

Coenzyme Q biochemistry and biosynthesis.

Coenzyme Q (CoQ) is a remarkably hydrophobic, redox-active lipid that empowers diverse cellular processes. Although most known for shuttling electrons between mitochondrial electron transport chain (ETC) complexes, the roles for CoQ are far more wide-reaching and ever-expanding. CoQ serves as a conduit for electrons from myriad pathways to enter the ETC, acts as a cofactor for biosynthetic and catabolic reactions, detoxifies damaging lipid species, and engages in cellular signaling and oxygen sensing. Many open questions remain regarding the biosynthesis, transport, and metabolism of CoQ, which hinders our ability to treat human CoQ deficiency. Here, we recount progress in filling these knowledge gaps, highlight unanswered questions, and underscore the need for novel tools to enable discoveries and improve the treatment of CoQ-related diseases.