The crucial role of lipids in cell, tissue and organ physiology is demonstrated by a large number of genetic studies and by many human diseases that involve the disruption of lipid metabolic enzymes and pathways. Examples of such diseases include cancer, diabetes, as well as neurodegenerative and infectious diseases. So far, the explosion of information in the fields of genomics and proteomics has not been matched by a corresponding advancement of knowledge in the field of lipids, which is largely due to the complexity of lipids and the lack of powerful tools for their analysis. Novel analytical approaches--in particular, liquid chromatography and mass spectrometry--for systems-level analysis of lipids and their interacting partners (lipidomics) now make this field a promising area of biomedical research, with a variety of applications in drug and biomarker development.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that is essential for normal brain growth and cognitive function. Consistent with its importance in the brain, DHA is highly enriched in brain phospholipids. Despite being an abundant fatty acid in brain phospholipids, DHA cannot be de novo synthesized in brain and must be imported across the blood-brain barrier, but mechanisms for DHA uptake in brain have remained enigmatic. Here we identify a member of the major facilitator superfamily--Mfsd2a (previously an orphan transporter)--as the major transporter for DHA uptake into brain. Mfsd2a is found to be expressed exclusively in endothelium of the blood-brain barrier of micro-vessels. Lipidomic analysis indicates that Mfsd2a-deficient (Mfsd2a-knockout) mice show markedly reduced levels of DHA in brain accompanied by neuronal cell loss in hippocampus and cerebellum, as well as cognitive deficits and severe anxiety, and microcephaly. Unexpectedly, cell-based studies indicate that Mfsd2a transports DHA in the form of lysophosphatidylcholine (LPC), but not unesterified fatty acid, in a sodium-dependent manner. Notably, Mfsd2a transports common plasma LPCs carrying long-chain fatty acids such LPC oleate and LPC palmitate, but not LPCs with less than a 14-carbon acyl chain. Moreover, we determine that the phosphor-zwitterionic headgroup of LPC is critical for transport. Importantly, Mfsd2a-knockout mice have markedly reduced uptake of labelled LPC DHA, and other LPCs, from plasma into brain, demonstrating that Mfsd2a is required for brain uptake of DHA. Our findings reveal an unexpected essential physiological role of plasma-derived LPCs in brain growth and function.

Lipid droplets (LDs) are emerging cellular organelles that are of crucial importance in cell biology and human diseases. In this study, we present our screen of ∼4,700 Saccharomyces cerevisiae mutants for abnormalities in the number and morphology of LDs; we identify 17 fld (few LDs) and 116 mld (many LDs) mutants. One of the fld mutants (fld1) is caused by the deletion of YLR404W, a previously uncharacterized open reading frame. Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1Δ cells demonstrate significantly enhanced fusion activities both in vivo and in vitro. Interestingly, the expression of human seipin, whose mutant forms are associated with Berardinelli-Seip congenital lipodystrophy and motoneuron disorders, rescues LD-associated defects in fld1Δ cells. Lipid profiling reveals alterations in acyl chain compositions of major phospholipids in fld1Δ cells. These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

SummaryRecent studies suggest that the sterol metabolic network participates in the interferon (IFN) antiviral response. However, the molecular mechanisms linking IFN with the sterol network and the identity of sterol mediators remain unknown. Here we report a cellular antiviral role for macrophage production of 25-hydroxycholesterol (cholest-5-en-3β,25-diol, 25HC) as a component of the sterol metabolic network linked to the IFN response via Stat1. By utilizing quantitative metabolome profiling of all naturally occurring oxysterols upon infection or IFN-stimulation, we reveal 25HC as the only macrophage-synthesized and -secreted oxysterol. We show that 25HC can act at multiple levels as a potent paracrine inhibitor of viral infection for a broad range of viruses. We also demonstrate, using transcriptional regulatory-network analyses, genetic interventions and chromatin immunoprecipitation experiments that Stat1 directly coupled Ch25h regulation to IFN in macrophages. Our studies describe a physiological role for 25HC as a sterol-lipid effector of an innate immune pathway.

Growing evidence suggests that close appositions between the endoplasmic reticulum (ER) and other membranes, including appositions with the plasma membrane (PM), mediate exchange of lipids between the two bilayers. The mechanisms of such exchange, which allows lipid transfer independently of vesicular transport, remain poorly understood. The presence of an SMP (synaptotagmin-like-mitochondrial-lipid binding protein) domain, a proposed lipid binding module, in several proteins localized at membrane contact sites raised the possibility that such domains may be implicated in lipid transport1,2. SMP-containing proteins include components of the ERMES complex, an ER-mitochondrial tether3, and the Extended-Synaptotagmins/tricalbins, which are ER-PM tethers4-6. Here we present at 2.44 Å resolution the crystal structure of a fragment of Extended-Synaptotagmin 2 (E-Syt2), including an SMP domain and two adjacent C2 domains. The SMP domain has a beta-barrel structure like protein modules in the TULIP superfamily. It dimerizes to form a ~90 Å long cylinder traversed by a channel lined entirely with hydrophobic residues, with the two C2A-C2B fragments forming arched structures flexibly linked to the SMP domain. Importantly, structural analysis complemented by mass spectrometry revealed the presence of glycerophospholipids in the E-Syt2 SMP channel, indicating a direct role for E-Syts in lipid transport. These findings provide strong evidence for a role of SMP domain containing proteins in the control of lipid transfer at membrane contact sites and have broad implication beyond the field of ER to PM appositions.

Lipid droplets (LDs) are important cellular organelles that govern the storage and turnover of lipids. Little is known about how the size of LDs is controlled, although LDs of diverse sizes have been observed in different tissues and under different (patho)physiological conditions. Recent studies have indicated that the size of LDs may influence adipogenesis, the rate of lipolysis and the oxidation of fatty acids. Here, a genome-wide screen identifies ten yeast mutants producing “supersized” LDs that are up to 50 times the volume of those in wild-type cells. The mutated genes include: FLD1, which encodes a homologue of mammalian seipin; five genes (CDS1, INO2, INO4, CHO2, and OPI3) that are known to regulate phospholipid metabolism; two genes (CKB1 and CKB2) encoding subunits of the casein kinase 2; and two genes (MRPS35 and RTC2) of unknown function. Biochemical and genetic analyses reveal that a common feature of these mutants is an increase in the level of cellular phosphatidic acid (PA). Results from in vivo and in vitro analyses indicate that PA may facilitate the coalescence of contacting LDs, resulting in the formation of “supersized” LDs. In summary, our results provide important insights into how the size of LDs is determined and identify novel gene products that regulate phospholipid metabolism.

SUMMARY Lipid composition can differ widely among organelles and even between leaflets of a membrane. Lipid homeostasis is critical because disequilibrium can have disease outcomes. Despite their importance, mechanisms maintaining lipid homeostasis remain poorly understood. Here, we establish a model system to study the global effects of lipid imbalance. Quantitative lipid profiling was integral to monitor changes to lipid composition and for system validation. Applying global transcriptional and proteomic analyses, a dramatically altered biochemical landscape was revealed from adaptive cells. The resulting composite regulation we term the “membrane stress response” (MSR) confers compensation, not through restoration of lipid composition, but by remodeling the protein homeostasis network. To validate its physiological significance, we analyzed the unfolded protein response (UPR), one facet of the MSR and a key regulator of protein homeostasis. We demonstrate that the UPR maintains protein biogenesis, quality control, and membrane integrity—functions otherwise lethally compromised in lipid dysregulated cells.

Phosphate is an essential nutrient for plant viability. It is well-established that phosphate starvation triggers membrane lipid remodeling, a process that converts significant portion of phospholipids to non-phosphorus-containing galactolipids. This remodeling is mediated by either phospholipase C (PLC) or phospholipase D (PLD) in combination with phosphatidate phosphatase (PAP). Two PLC genes, NPC4 and NPC5, and PLD genes, PLD1 and PLD2, are shown to be involved in the remodeling. However, gene knockout studies show that none of them plays decisive roles in the remodeling. Thus, although this phenomenon is widely observed among plants, the key enzyme(s) responsible for the lipid remodeling in a whole plant body is unknown; therefore, the physiological significance of this conversion process has remained to be elucidated. We herein focused on PAP as a key enzyme for this adaptation, and identified Arabidopsis lipin homologs, AtPAH1 and AtPAH2, that encode the PAPs involved in galactolipid biosynthesis. Double mutant pah1pah2 plants had decreased phosphatidic acid hydrolysis, thus affecting the eukaryotic pathway of galactolipid synthesis. Upon phosphate starvation, pah1pah2 plants were severely impaired in growth and membrane lipid remodeling. These results indicate that PAH1 and PAH2 are the PAP responsible for the eukaryotic pathway of galactolipid synthesis, and the membrane lipid remodeling mediated by these two enzymes is an essential adaptation mechanism to cope with phosphate starvation.galactolipids ͉ phosphatidic acid ͉ phosphatidic acid phosphatase