Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila.

Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. We now find that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. This study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts.

Metabolic priming by multiple enzyme systems supports glycolysis, HIF1α stabilisation, and human cancer cell survival in early hypoxia.

Adaptation to chronic hypoxia occurs through changes in protein expression, which are controlled by hypoxia-inducible factor 1α (HIF1α) and are necessary for cancer cell survival. However, the mechanisms that enable cancer cells to adapt in early hypoxia, before the HIF1α-mediated transcription programme is fully established, remain poorly understood. Here we show in human breast cancer cells, that within 3 h of hypoxia exposure, glycolytic flux increases in a HIF1α-independent manner but is limited by NAD+ availability. Glycolytic ATP maintenance and cell survival in early hypoxia rely on reserve lactate dehydrogenase A capacity as well as the activity of glutamate-oxoglutarate transaminase 1 (GOT1), an enzyme that fuels malate dehydrogenase 1 (MDH1)-derived NAD+. In addition, GOT1 maintains low α-ketoglutarate levels, thereby limiting prolyl hydroxylase activity to promote HIF1α stabilisation in early hypoxia and enable robust HIF1α target gene expression in later hypoxia. Our findings reveal that, in normoxia, multiple enzyme systems maintain cells in a primed state ready to support increased glycolysis and HIF1α stabilisation upon oxygen limitation, until other adaptive processes that require more time are fully established.

Protein Kinase C-ß Dictates B Cell Fate by Regulating Mitochondrial Remodeling, Metabolic Reprogramming, and Heme Biosynthesis.

PKCß-null (Prkcb-/-) mice are severely immunodeficient. Here we show that mice whose B cells lack PKCß failed to form germinal centers and plasma cells, which undermined affinity maturation and antibody production in response to immunization. Moreover, these mice failed to develop plasma cells in response to viral infection. At the cellular level, we have shown that Prkcb-/- B cells exhibited defective antigen polarization and mTORC1 signaling. While altered antigen polarization impaired antigen presentation and likely restricted the potential of GC development, defective mTORC1 signaling impaired metabolic reprogramming, mitochondrial remodeling, and heme biosynthesis in these cells, which altogether overwhelmingly opposed plasma cell differentiation. Taken together, our study reveals mechanistic insights into the function of PKCß as a key regulator of B cell polarity and metabolic reprogramming that instructs B cell fate.

A malaria parasite phospholipase facilitates efficient asexual blood stage egress.

Malaria parasite release (egress) from host red blood cells involves parasite-mediated membrane poration and rupture, thought to involve membrane-lytic effector molecules such as perforin-like proteins and/or phospholipases. With the aim of identifying these effectors, we disrupted the expression of two Plasmodium falciparum perforin-like proteins simultaneously and showed that they have no essential roles during blood stage egress. Proteomic profiling of parasite proteins discharged into the parasitophorous vacuole (PV) just prior to egress detected the presence in the PV of a lecithin:cholesterol acyltransferase (LCAT; PF3D7_0629300). Conditional ablation of LCAT resulted in abnormal egress and a reduced replication rate. Lipidomic profiles of LCAT-null parasites showed drastic changes in several phosphatidylserine and acylphosphatidylglycerol species during egress. We thus show that, in addition to its previously demonstrated role in liver stage merozoite egress, LCAT is required to facilitate efficient egress in asexual blood stage malaria parasites.

Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis.

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes.

Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling.

Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a 13C labeling approach, called 13C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples and showed the distinct kinetics of the N6-methyladenosine (m6A) versus 7-methylguanosine (m7G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m62A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under nonstationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs.

Author Correction: MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium-dependent protein kinase 3.

Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium-dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3-dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5-3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5-3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non-phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5-3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle.

MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG.

α-Ketoglutarate (αKG) is a key node in many important metabolic pathways. The αKG analog N-oxalylglycine (NOG) and its cell-permeable prodrug dimethyloxalylglycine (DMOG) are extensively used to inhibit αKG-dependent dioxygenases. However, whether NOG interference with other αKG-dependent processes contributes to its mode of action remains poorly understood. Here we show that, in aqueous solutions, DMOG is rapidly hydrolyzed, yielding methyloxalylglycine (MOG). MOG elicits cytotoxicity in a manner that depends on its transport by monocarboxylate transporter 2 (MCT2) and is associated with decreased glutamine-derived tricarboxylic acid-cycle flux, suppressed mitochondrial respiration and decreased ATP production. MCT2-facilitated entry of MOG into cells leads to sufficiently high concentrations of NOG to inhibit multiple enzymes in glutamine metabolism, including glutamate dehydrogenase. These findings reveal that MCT2 dictates the mode of action of NOG by determining its intracellular concentration and have important implications for the use of (D)MOG in studying αKG-dependent signaling and metabolism.

Cryogenic OrbiSIMS Localizes Semi-Volatile Molecules in Biological Tissues.

OrbiSIMS is a recently developed instrument for label-free imaging of chemicals with micron spatial resolution and high mass resolution. We report a cryogenic workflow for OrbiSIMS (Cryo-OrbiSIMS) that improves chemical detection of lipids and other biomolecules in tissues. Cryo-OrbiSIMS boosts ionization yield and decreases ion-beam induced fragmentation, greatly improving the detection of biomolecules such as triacylglycerides. It also increases chemical coverage to include molecules with intermediate or high vapor pressures, such as free fatty acids and semi-volatile organic compounds (SVOCs). We find that Cryo-OrbiSIMS reveals the hitherto unknown localization patterns of SVOCs with high spatial and chemical resolution in diverse plant, animal, and human tissues. We also show that Cryo-OrbiSIMS can be combined with genetic analysis to identify enzymes regulating SVOC metabolism. Cryo-OrbiSIMS is applicable to high resolution imaging of a wide variety of non-volatile and semi-volatile molecules across many areas of biomedicine.

Metabolic reprogramming and Notch activity distinguish between non-small cell lung cancer subtypes.

BACKGROUND: Previous studies suggested that the metabolism is differently reprogrammed in the major subtypes of non-small cell lung cancer (NSCLC), squamous cell carcinomas (SCC) and adenocarcinomas (AdC). However, a comprehensive analysis of this differential metabolic reprogramming is lacking. METHODS: Publicly available gene expression data from human lung cancer samples and cell lines were analysed. Stable isotope resolved metabolomics were performed on SCC and ADC tumours in human patients and in freshly resected tumour slices. RESULTS: Analysis of multiple transcriptomics data from human samples identified a SCC-distinguishing enzyme gene signature. SCC tumours from patients infused with [U-13C]-glucose and SCC tissue slices incubated with stable isotope tracers demonstrated differential glucose and glutamine catabolism compared to AdCs or non-cancerous lung, confirming increased activity through pathways defined by the SCC metabolic gene signature. Furthermore, the upregulation of Notch target genes was a distinguishing feature of SCCs, which correlated with the metabolic signature. Notch and MYC-driven murine lung tumours recapitulated the SCC-distinguishing metabolic reprogramming. However, the differences between SCCs and AdCs disappear in established cell lines in 2D culture. CONCLUSIONS: Our data emphasise the importance of studying lung cancer metabolism in vivo. They also highlight potential targets for therapeutic intervention in SCC patients including differentially expressed enzymes that catalyse reactions in glycolysis, glutamine catabolism, serine, nucleotide and glutathione biosynthesis.

Metabolic oscillations on the circadian time scale in Drosophila cells lacking clock genes.

Circadian rhythms are cell-autonomous biological oscillations with a period of about 24 h. Current models propose that transcriptional feedback loops are the primary mechanism for the generation of circadian oscillations. Within this framework, Drosophila S2 cells are regarded as "non-rhythmic" cells, as they do not express several canonical circadian components. Using an unbiased multi-omics approach, we made the surprising discovery that Drosophila S2 cells do in fact display widespread daily rhythms. Transcriptomics and proteomics analyses revealed that hundreds of genes and their products, and in particular metabolic enzymes, are rhythmically expressed in a 24-h cycle. Metabolomics analyses extended these findings and demonstrate that central carbon metabolism and amino acid metabolism are core metabolic pathways driven by protein rhythms. We thus demonstrate that 24-h metabolic oscillations, coupled to gene and protein cycles, take place in nucleated cells without the contribution of any known circadian regulators. These results therefore suggest a reconsideration of existing models of the clockwork in Drosophila and other eukaryotic systems.

Reduced cholesterol levels impair Smoothened activation in Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a common autosomal-recessive disorder that results from mutations in the gene encoding the cholesterol biosynthetic enzyme 7-dehydrocholesterol reductase (DHCR7). Impaired DHCR7 function is associated with a spectrum of congenital malformations, intellectual impairment, epileptiform activity and autism spectrum disorder. Biochemically, there is a deficit in cholesterol and an accumulation of its metabolic precursor 7-dehydrocholesterol (7DHC) in developing tissues. Morphological abnormalities in SLOS resemble those seen in congenital Sonic Hedgehog (SHH)-deficient conditions, leading to the proposal that the pathogenesis of SLOS is mediated by aberrant SHH signalling. SHH signalling is transduced through the transmembrane protein Smoothened (SMO), which localizes to the primary cilium of a cell on activation and is both positively and negatively regulated by sterol molecules derived from cholesterol biosynthesis. One proposed mechanism of SLOS involves SMO dysregulation by altered sterol levels, but the salient sterol species has not been identified. Here, we clarify the relationship between disrupted cholesterol metabolism and reduced SHH signalling in SLOS by modelling the disorder in vitro. Our results indicate that a deficit in cholesterol, as opposed to an accumulation of 7DHC, impairs SMO activation and its localization to the primary cilium.

Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments.

Malaria parasites (Plasmodium spp.) encounter markedly different (nutritional) environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA) metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design.

Apicoplast-Localized Lysophosphatidic Acid Precursor Assembly Is Required for Bulk Phospholipid Synthesis in Toxoplasma gondii and Relies on an Algal/Plant-Like Glycerol 3-Phosphate Acyltransferase.

Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii.

Characterization of the Plasmodium falciparum and P. berghei glycerol 3-phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast.

Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti-malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3-phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N-terminal targeting sequence to GFP and 3' tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site-directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3-phosphate acyltransferase in malaria parasites.

Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate.

Organelle gain through endosymbiosis has been integral to the origin and diversification of eukaryotes, and, once gained, plastids and mitochondria seem seldom lost. Indeed, discovery of nonphotosynthetic plastids in many eukaryotes--notably, the apicoplast in apicomplexan parasites such as the malaria pathogen Plasmodium--highlights the essential metabolic functions performed by plastids beyond photosynthesis. Once a cell becomes reliant on these ancillary functions, organelle dependence is apparently difficult to overcome. Previous examples of endosymbiotic organelle loss (either mitochondria or plastids), which have been invoked to explain the origin of eukaryotic diversity, have subsequently been recognized as organelle reduction to cryptic forms, such as mitosomes and apicoplasts. Integration of these ancient symbionts with their hosts has been too well developed to reverse. Here, we provide evidence that the dinoflagellate Hematodinium sp., a marine parasite of crustaceans, represents a rare case of endosymbiotic organelle loss by the elimination of the plastid. Extensive RNA and genomic sequencing data provide no evidence for a plastid organelle, but, rather, reveal a metabolic decoupling from known plastid functions that typically impede organelle loss. This independence has been achieved through retention of ancestral anabolic pathways, enzyme relocation from the plastid to the cytosol, and metabolic scavenging from the parasite's host. Hematodinium sp. thus represents a further dimension of endosymbiosis--life after the organelle.

The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell.

Apicomplexa are parasitic protozoa that cause important human diseases including malaria, cryptosporidiosis and toxoplasmosis. The replication of these parasites within their target host cell is dependent on both salvage as well as de novo synthesis of fatty acids. In Toxoplasma gondii, fatty acid synthesis via the apicoplast-localized FASII is essential for pathogenesis, while the role of two other fatty acid biosynthetic complexes remains unclear. Here, we demonstrate that the ER-localized fatty acid elongation (ELO) complexes are essential for parasite growth. Conditional knockdown of the nonredundant hydroxyacyl-CoA dehydratase and enoyl-CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth. (13) C-glucose and (13) C-acetate labeling and comprehensive lipidomic analyses of these mutants showed a selective defect in synthesis of unsaturated long and very long-chain fatty acids (LCFAs and VLCFAs) and depletion of phosphatidylinositol and phosphatidylethanolamine species containing unsaturated LCFAs and VLCFAs. This requirement for ELO pathway was bypassed by supplementing the media with specific fatty acids, indicating active but inefficient import of host fatty acids. Our experiments highlight a gap between the fatty acid needs of the parasite and availability of specific fatty acids in the host cell that the parasite has to close using a dedicated synthesis and modification pathway.

Metabolomics-Based Screening of the Malaria Box Reveals both Novel and Established Mechanisms of Action.

High-throughput phenotypic screening of chemical libraries has resulted in the identification of thousands of compounds with potent antimalarial activity, although in most cases, the mechanism(s) of action of these compounds remains unknown. Here we have investigated the mode of action of 90 antimalarial compounds derived from the Malaria Box collection using high-coverage, untargeted metabolomics analysis. Approximately half of the tested compounds induced significant metabolic perturbations in in vitro cultures of Plasmodium falciparum In most cases, the metabolic profiles were highly correlated with known antimalarials, in particular artemisinin, the 4-aminoquinolines, or atovaquone. Select Malaria Box compounds also induced changes in intermediates in essential metabolic pathways, such as isoprenoid biosynthesis (i.e., 2-C-methyl-d-erythritol 2,4-cyclodiphosphate) and linolenic acid metabolism (i.e., traumatic acid). This study provides a comprehensive database of the metabolic perturbations induced by chemically diverse inhibitors and highlights the utility of metabolomics for triaging new lead compounds and defining specific modes of action, which will assist with the development and optimization of new antimalarial drugs.