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Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase–cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a ‘folate trap’. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.

European canker, caused by the necrotrophic fungus Neonectria ditissima, is the most serious disease in apple production in Sweden. The disease is favored by a relatively cool and rainy climate. The canker damages have a significant economic impact due to reduced bearing surface and increased orchard management costs. The possibilities for chemical and biological control are very limited. Therefore, directed breeding for new resistant cultivars is urgently needed. Knowledge of inheritance of canker resistance and understanding of molecular mechanisms involved in resistant and susceptible responses to fungal attacks would facilitate breeding. In this study, we evaluated the tempo-spatial differences in plant-pathogen interactions in a set of partially resistant and susceptible cultivars by conducting metabolomic and lipidomic analyses. The major trends in metabolomics and lipidomic profiles were common among cultivars, irrespective of the degree of susceptibility. Several metabolites and lipids varied with time point and cultivar under N. ditissima infection. Putative key metabolites such as suberic acid and jasmonic acid were upregulated in all cultivars upon infection. Additionally, several lipids exhibited changes 30 to 45 days post-inoculation. Thus, the approach used seems to have resulted in a rich data set to be further analyzed in light of ongoing QTL-mapping efforts.

R2-like ligand-binding oxidase (R2lox) is a ferritin-like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross-link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry-based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.

Mitophagy removes defective mitochondria via lysosomal elimination. Increased mitophagy coincides with metabolic reprogramming, yet it remains unknown whether mitophagy is a cause or consequence of such state changes. The signalling pathways that integrate with mitophagy to sustain cell and tissue integrity also remain poorly defined. We performed temporal metabolomics on mammalian cells treated with deferiprone, a therapeutic iron chelator that stimulates PINK1/PARKIN-independent mitophagy. Iron depletion profoundly rewired the metabolome, hallmarked by remodelling of lipid metabolism within minutes of treatment. DGAT1-dependent lipid droplet biosynthesis occurred several hours before mitochondrial clearance, with lipid droplets bordering mitochondria upon iron chelation. We demonstrate that DGAT1 inhibition restricts mitophagy in vitro, with impaired lysosomal homeostasis and cell viability. Importantly, genetic depletion of DGAT1 in vivo significantly impaired neuronal mitophagy and locomotor function in Drosophila. Our data define iron depletion as a potent signal that rapidly reshapes metabolism and establishes an unexpected synergy between lipid homeostasis and mitophagy that safeguards cell and tissue integrity.

Caveolae are bulb-shaped invaginations of the plasma membrane (PM) that undergo scission and fusion at the cell surface and are enriched in specific lipids. However, the influence of lipid composition on caveolae surface stability is not well described or understood. Accordingly, we inserted specific lipids into the cell PM via membrane fusion and studied their acute effects on caveolae dynamics. We demonstrate that sphingomyelin stabilizes caveolae to the cell surface, whereas cholesterol and glycosphingolipids drive caveolae scission from the PM. Although all three lipids accumulated specifically in caveolae, cholesterol and sphingomyelin were actively sequestered, whereas glycosphingolipids diffused freely. The ATPase EHD2 restricts lipid diffusion and counteracts lipid-induced scission. We propose that specific lipid accumulation in caveolae generates an intrinsically unstable domain prone to scission if not restrained by EHD2 at the caveolae neck. This work provides a mechanistic link between caveolae and their ability to sense the PM lipid composition.

This study investigates the role of the circadian clock in the regulation of gibberellin (GA) metabolism and growth in hybrid aspen (Populus tremula x P. tremuloides (Ptt)). We revealed a conserved function of the clock homolog PttEARLY BIRD1 (PttEBI1), but also implicated its rolein controlling tree growth. GA metabolite profiling and transcriptomic analysis in hybrid aspenlines with modulated expression of PttEBI1 or the core clock homologs PttLATE ELONGATED HYPOCOTYLs (PttLHYs) revealed significant changes in GA metabolism. These alterations werelinked to the differential expression of PttGA2ox8, a gene encoding an enzyme with both GA2-oxidase and GA20-oxidase activities. Our results indicate that modifications to circadian clockcomponents can significantly influence both GA metabolism and tree growth, providing potential strategies for improving tree biomass production.