Seipin transmembrane segments critically function in triglyceride nucleation and lipid droplet budding from the membrane

ABSTRACTLipid droplets (LDs) are organelles formed in the endoplasmic reticulum (ER) to store triacylglycerol (TG) and sterol esters. The ER protein seipin is key for LD biogenesis. Seipin forms a cage-like structure, with each seipin monomer containing a conserved hydrophobic helix (HH) and two transmembrane (TM) segments. How the different parts of seipin function in TG nucleation and LD budding is poorly understood. Here, we utilized molecular dynamics simulations of human seipin, along with cell-based experiments, to study seipin’s functions in protein-lipid interactions, lipid diffusion, and LD maturation. All-atom (AA) simulations indicate that most seipin TM segment residues located in the phospholipid (PL) tail region of the bilayer attract TG. We also find seipin TM segments control lipid diffusion and permeation into the protein complex. Simulating larger, growing LDs with coarse-grained (CG) models, we find that the seipin TM segments form a constricted neck structure to facilitate conversion of a flat oil lens into a budding LD. Using cell experiments and simulations, we also show that conserved, positively charged residues at the end of seipin’s TM segments affect LD maturation. We propose a model in which seipin TM segments critically function in TG nucleation and LD growth.

Comparative Metabolomics Analysis Reveals Sterols and Sphingolipids Play a Role in Cotton Fiber Cell Initiation

Cotton fiber is a seed trichome that protrudes from the outer epidermis of cotton ovule on the day of anthesis (0 day past anthesis, 0 DPA). The initial number and timing of fiber cells are closely related to fiber yield and quality. However, the mechanism underlying fiber initiation is still unclear. Here, we detected and compared the contents and compositions of sphingolipids and sterols in 0 DPA ovules of Xuzhou142 lintless-fuzzless mutants (Xufl) and Xinxiangxiaoji lintless-fuzzless mutants (Xinfl) and upland cotton wild-type Xuzhou142 (XuFL). Nine classes of sphingolipids and sixty-six sphingolipid molecular species were detected in wild-type and mutants. Compared with the wild type, the contents of Sphingosine-1-phosphate (S1P), Sphingosine (Sph), Glucosylceramide (GluCer), and Glycosyl-inositol-phospho-ceramides (GIPC) were decreased in the mutants, while the contents of Ceramide (Cer) were increased. Detail, the contents of two Cer molecular species, d18:1/22:0 and d18:1/24:0, and two Phyto-Cer molecular species, t18:0/22:0 and t18:0/h22:1 were significantly increased, while the contents of all GluCer and GIPC molecular species were decreased. Consistent with this result, the expression levels of seven genes involved in GluCer and GIPC synthesis were decreased in the mutants. Furthermore, exogenous application of a specific inhibitor of GluCer synthase, PDMP (1-phenyl-2-decanoylamino-3-morpholino-1-propanol), in ovule culture system, significantly inhibited the initiation of cotton fiber cells. In addition, five sterols and four sterol esters were detected in wild-type and mutant ovules. Compared with the wild type, the contents of total sterol were not significantly changed. While the contents of stigmasterol and campesterol were significantly increased, the contents of cholesterol were significantly decreased, and the contents of total sterol esters were significantly increased. In particular, the contents of campesterol esters and stigmasterol esters increased significantly in the two mutants. Consistently, the expression levels of some sterol synthase genes and sterol ester synthase genes were also changed in the two mutants. These results suggested that sphingolipids and sterols might have some roles in the initiation of fiber cells. Our results provided a novel insight into the regulatory mechanism of fiber cell initiation.

Liquid-crystalline lipid phase transitions in lipid droplets selectively remodel the LD proteome

Lipid droplets (LDs) are reservoirs for triglycerides (TGs) and sterol-esters (SEs). How lipids are organized within LDs and influence the LD proteome remains unclear. Using in situ cryo-electron tomography, we show that glucose restriction triggers lipid phase transitions within LDs generating liquid-crystalline lattices inside them. Mechanistically, this requires TG lipolysis, which alters LD neutral lipid composition and promotes SE transition to a liquid-crystalline phase. Fluorescence imaging and proteomics further reveal that LD liquid-crystalline lattices selectively remodel the LD proteome. Some canonical LD proteins including Erg6 re-localize to the ER network, whereas others remain on LDs. Model peptide LiveDrop also redistributes from LDs to the ER, suggesting liquid-crystalline-phases influence ER-LD inter-organelle transport. Proteomics also indicates glucose restriction elevates peroxisome lipid oxidation, suggesting TG mobilization provides fatty acids for cellular energetics. This suggests glucose restriction drives TG mobilization, which alters the phase properties of LD lipids and selectively remodels the LD proteome.

Combined Biosynthetic Pathway Engineering and Storage Pool Expansion for High-Level Production of Ergosterol in Industrial Saccharomyces cerevisiae

Ergosterol, a terpenoid compound produced by fungi, is an economically important metabolite serving as the direct precursor of steroid drugs. Herein, ergsosterol biosynthetic pathway modification combined with storage capacity enhancement was proposed to synergistically improve the production of ergosterol in Saccharomyces cerevisiae. S. cerevisiae strain S1 accumulated the highest amount of ergosterol [7.8 mg/g dry cell weight (DCW)] among the wild-type yeast strains tested and was first selected as the host for subsequent metabolic engineering studies. Then, the push and pull of ergosterol biosynthesis were engineered to increase the metabolic flux, overexpression of the sterol acyltransferase gene ARE2 increased ergosterol content to 10 mg/g DCW and additional overexpression of a global regulatory factor allele (UPC2-1) increased the ergosterol content to 16.7 mg/g DCW. Furthermore, considering the hydrophobicity sterol esters and accumulation in lipid droplets, the fatty acid biosynthetic pathway was enhanced to expand the storage pool for ergosterol. Overexpression of ACC1 coding for the acetyl-CoA carboxylase increased ergosterol content from 16.7 to 20.7 mg/g DCW. To address growth inhibition resulted from premature accumulation of ergosterol, auto-inducible promoters were employed to dynamically control the expression of ARE2, UPC2-1, and ACC1. Consequently, better cell growth led to an increase of ergosterol content to 40.6 mg/g DCW, which is 4.2-fold higher than that of the starting strain. Finally, a two-stage feeding strategy was employed for high-density cell fermentation, with an ergosterol yield of 2986.7 mg/L and content of 29.5 mg/g DCW. This study provided an effective approach for the production of ergosterol and other related terpenoid molecules.

Lipidomic Signatures for Colorectal Cancer Diagnosis and Progression Using UPLC-QTOF-ESI+MS

Metabolomics coupled with bioinformatics may identify relevant biomolecules such as putative biomarkers of specific metabolic pathways related to colorectal diagnosis, classification and prognosis. This study performed an integrated metabolomic profiling of blood serum from 25 colorectal cancer (CRC) cases previously classified (Stage I to IV) compared with 16 controls (disease-free, non-CRC patients), using high-performance liquid chromatography and mass spectrometry (UPLC-QTOF-ESI+ MS). More than 400 metabolites were separated and identified, then all data were processed by the advanced Metaboanalyst 5.0 online software, using multi- and univariate analysis, including specificity/sensitivity relationships (area under the curve (AUC) values), enrichment and pathway analysis, identifying the specific pathways affected by cancer progression in the different stages. Several sub-classes of lipids including phosphatidylglycerols (phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and PAs), fatty acids and sterol esters as well as ceramides confirmed the “lipogenic phenotype” specific to CRC development, namely the upregulated lipogenesis associated with tumor progression. Both multivariate and univariate bioinformatics confirmed the relevance of some putative lipid biomarkers to be responsible for the altered metabolic pathways in colorectal cancer.

CHEMICAL RESEARCH AND BIOLOGICAL ACTIVITY OF PLANTS OF THE GENUS ATRAPHAXIS (A. SPINOSA)

In this article identifies new sources of obtaining biological substances from plants of the genus Atraphaxis (A. spinosa) prepared in the Almaty region. According to well - known methods, the analysis of indicators and standards of raw material quality-humidity, total ash, sulphate ash, insoluble ash in 10% hydrochloric acid-was developed and carried out. Micro- and macroelements determined by atomic absorption spectroscopy. Analysis of the elemental composition shows that iron predominates from microelements, and sodium, potassium and calcium from macronutrients. Conditions for obtaining a biologically active complex from the aboveground part of Atraphaxis spinosa developed for the first time. The optimal conditions for obtaining the complex are extractant – 50% ethanol, the ratio of extractant and raw materials – 1:8, double extraction time – 48 hours, temperature – 22 - 26 °C. The lipophilic composition identified by chromatography-mass spectroscopy. Since lipophilic fractions of plant samples include such classes of compounds as fatty acids; mono-; di-; triglycerides, phospholipids, sterols, Sterol esters, glycolipids, fat-soluble vitamins, they can considered not only as nutritional products, but also as possible pharmacological agents. The content of lipophilic components – 26 organic compounds-was determined. It found that A. spinosa contains a large amount of di - (2-ethylhexyl) phthalate (54.66%) and β-sitosterol (13.11%). A complex study of plant resources as medicinal raw materials provides for the chemical study of biologically active substances and biological screening of extracts and individual compounds obtained from plants. In most cases, the extract showed a wide range of antibacterial activity against the used strains of microorganisms.

Larval energetics of the Sydney rock oyster Saccostrea glomerata and Pacific oyster Magallana gigas

Larvae are a critical dispersal stage of marine invertebrates, and their survival depends on nutrition and energetics. This study compared the size, survival, metabolic rate and egg and larval lipid class profiles of larvae of the endemic Sydney rock oyster Saccostrea glomerata and the invasive Pacific oyster Magallana gigas through a period of starvation for 5 and 9 d after fertilisation. Starved larvae grew without food until 5 d of age, at which point they stopped developing, but resumed growth when fed. Egg lipids profiles comprised 78.1 and 74.5% triacylglycerol for M. gigas and S. glomerata respectively. When fed, larvae of M. gigas were significantly larger in size and had faster growth and similar survival compared to S. glomerata. When starved, larvae of M. gigas and S. glomerata grew at similar rates, and there was a trend for lower survival of M. gigas. Larval endogenous lipid reserves were deleted in the first 24 h. Larvae of M. gigas had more total lipids and comparatively more diacylglycerols, monoacylglycerols, phospholipids and cholesterol, whereas S. glomerata had more diacylglycerols and produced sterol esters. Starvation altered the patterns of lipid assimilation, and metabolic rates of larvae of M. gigas and S. glomerata differed over time. When starved, S. glomerata larvae had greater capacity to cope with starvation compared to M. gigas, perhaps due to an evolutionary history in oligotrophic estuaries. As the climate rapidly changes in this global climate-change hotspot, S. glomerata is likely to be negatively affected.

Changes in Lipid Composition of Streptomyces massasporeus CNMN-AC-06 Biomass after Long-Term Storage

Aim. The aim of the research was to determine changes in content and composition of Streptomyces massasporeus CNMN-Ac-06 strain biomass lipid complex during cultivation in various nutrient media after long-term storage. Methods. To obtain the inoculum, S. massasporeus CNMN-Ac-06 strain was cultivated in Dulaney medium. For biomass accumulation, inoculum was cultivated in M-I, SP-I and SP-III. The amount of biomass was determined on the 5th day of culture growth. Intracellular lipids were extracted from streptomycete biomass by Folch method modified in our laboratory. The qualitative and quantitative characteristic of lipid complex composition was determined by thin-layer chromatography. 10% solution of phosphomolybdic acid in ethanol was used as developer. The quantity of individual lipid fractions was determined by densitometry. Results. The studied strain was cultivated in three nutrient liquid media. Cultivation of the strain in M-I medium increases the biomass yield up to 11.53 g/l. In case of SP-III, on the contrary, at the beginning of research, the biomass yield was higher than after storage. The best result of the synthesis of total lipids was noted after SP-I medium use. Analysis of the quantity of main lipid fractions in the biomass showed that the maximum percentage of phospholipids was 12.15% after cultivation in SP-I medium. The amount of sterols in biomass was: in M-I medium – 8.96%, in SP-I medium – 12.15% and in SP-III medium – 14.17%. The smallest amount of mono- and diglycerides in the total lipids of the biomass of this strain was observed after cultivation in SP-III medium, sterol esters in SP-I medium, and waxes in M-I medium. The studies shown that the highest amount of biomass of the strain S. massasporeus CNMN-Ac-06 was noted after cultivation in nutrient medium M-I (11.53 g/l), and the maximum percentage of total lipids after cultivation in medium SP-I (15.85%). Conclusions. The experiments shown that in order to increase the biomass of S. massasporeus CNMN-Ac-06 strain, it is the best to cultivate this microorganism in complex SP-I medium. But, a significant amount of such physiologically important lipid fractions like phospholipids was obtained in SP-I medium and sterols in SP-I and SP-III media.