Exploring selection signatures in the divergence and evolution of lipid droplet (LD) associated genes in major oilseed crops.

BACKGROUND: Oil bodies or lipid droplets (LDs) in the cytosol are the subcellular storage compartments of seeds and the sites of lipid metabolism providing energy to the germinating seeds. Major LD-associated proteins are lipoxygenases, phospholipaseD, oleosins, TAG-lipases, steroleosins, caleosins and SEIPINs; involved in facilitating germination and enhancing peroxidation resulting in off-flavours. However, how natural selection is balancing contradictory processes in lipid-rich seeds remains evasive. The present study was aimed at the prediction of selection signatures among orthologous clades in major oilseeds and the correlation of selection effect with gene expression. RESULTS: The LD-associated genes from the major oil-bearing crops were analyzed to predict natural selection signatures in phylogenetically close-knit ortholog clusters to understand adaptive evolution. Positive selection was the major force driving the evolution and diversification of orthologs in a lineage-specific manner. Significant positive selection effects were found in 94 genes particularly in oleosin and TAG-lipases, purifying with excess of non-synonymous substitution in 44 genes while 35 genes were neutral to selection effects. No significant selection impact was noticed in Brassicaceae as against LOX genes of oil palm. A heavy load of deleterious mutations affecting selection signatures was detected in T-lineage oleosins and LOX genes of Arachis hypogaea. The T-lineage oleosin genes were involved in mainly anther, tapetum and anther wall morphogenesis. In Ricinus communis and Sesamum indicum > 85% of PLD genes were under selection whereas selection pressures were low in Brassica juncea and Helianthus annuus. Steroleosin, caleosin and SEIPINs with large roles in lipid droplet organization expressed mostly in seeds and were under considerable positive selection pressures. Expression divergence was evident among paralogs and homeologs with one gene attaining functional superiority compared to the other. The LOX gene Glyma.13g347500 associated with off-flavor was not expressed during germination, rather its paralog Glyma.13g347600 showed expression in Glycine max. PLD-α genes were expressed on all the tissues except the seed,δ genes in seed and meristem while ß and γ genes expressed in the leaf. CONCLUSIONS: The genes involved in seed germination and lipid metabolism were under strong positive selection, although species differences were discernable. The present study identifies suitable candidate genes enhancing seed oil content and germination wherein directional selection can become more fruitful.

Specific nasopharyngeal Corynebacterium strains serve as gatekeepers against SARS-CoV-2 infection.

The SARS-CoV-2 virus is still causing a worldwide problem. The virus settles primarily on the nasal mucosa, and the infection and its course depend on individual susceptibility. Our aim was to investigate the nasopharynx composition's role in the individual susceptibility. During the first phase of SARS-CoV-2 pandemic, nasopharyngeal microbiome samples of close contact unvaccinated patients were investigated by 16S rRNA analysis and by culturing. The whole genome of cultured Corynebacteria was sequenced. The relative expression of ACE2, TMPRSS2, and cathepsin L on Caco-2 cells and the strength of S1-ACE2 binding were determined in the presence of Corynebacteria. From 55 close contacts exposed to identical SARS-CoV-2 exposure, 26 patients became infected and 29 remained uninfected. The nasopharyngeal microbiome analysis showed significantly higher abundance of Corynebacteria in uninfected group. Corynebacterium accolens could be cultivated only from uninfected individuals and Corynebacterium propinquum from both infected and uninfected. Corynebacteria from uninfected patient significantly reduced the ACE2 and cathepsin L expression. C. accolens significantly reduced the TMPRSS2 expression compared to other Corynebacteria. Furthermore, Corynebacterium spp. weakened the binding of the S1-ACE2. Most C. accolens isolates harbored the TAG lipase LipS1 gene. Based on these results, the presence of Corynebacterium spp. in the nasopharyngeal microbiota, especially C. accolens strains, could reduce the individual susceptibility to SARS-CoV-2 infection by several mechanisms: by downregulation the ACE2, the TMPRSS2 receptors, and cathepsin L in the host; through the inhibition of S1-ACE2 binding; and lipase production. These results suggest the use of C. accolens strains as probiotics in the nasopharynx in the future.

DAGL-Beta Functions as a PUFA-Specific Triacylglycerol Lipase in Macrophages.

Here, we apply quantitative chemical proteomics and untargeted lipidomics to assign a polyunsaturated fatty acid (PUFA)-specific triacylglycerol (TAG) lipase activity for diacylglycerol lipase-beta (DAGLß) in macrophages. We demonstrate that DAGLß but not DAGLα is expressed and active in bone marrow-derived macrophages (BMDMs) as determined by activity-based protein profiling analysis of SILAC BMDMs. Genetic disruption of DAGLß resulted in accumulation of cellular TAGs composed of PUFA but not saturated/low unsaturated fatty acid counterparts, which is recapitulated in wild-type macrophages treated with a DAGLß-selective inhibitor. Biochemical assays with synthetic substrates confirm PUFA-TAGs as authentic DAGLß substrates. In summary, our findings identify DAGLß as a PUFA-specific TAG lipase in primary macrophages.

The protein-sparing effect of α-lipoic acid in juvenile grass carp, Ctenopharyngodon idellus: effects on lipolysis, fatty acid ß-oxidation and protein synthesis.

To investigate the protein-sparing effect of α-lipoic acid (LA), experimental fish (initial body weight: 18·99 (sd 1·82) g) were fed on a 0, 600 or 1200 mg/kg α-LA diet for 56 d, and hepatocytes were treated with 20 µm compound C, the inhibitor of AMP kinase α (AMPKα), treated for 30 min before α-LA treatment for 24 h. LA significantly decreased lipid content of the whole body and other tissues (P0·05). Consistent with results from the experiment in vitro, LA activated phosphorylation of AMPKα and notably increased the protein content of adipose TAG lipase in intraperitoneal fat, hepatopancreas and muscle in vivo (P<0·05). Meanwhile, LA significantly up-regulated the mRNA expression of genes involved in fatty acid ß-oxidation in the same three areas, and LA also obviously down-regulated the mRNA expression of genes involved in amino acid catabolism in muscle (P<0·05). Besides, it was observed that LA significantly activated the mammalian target of rapamycin (mTOR) pathway in muscle of experimental fish (P<0·05). LA could promote lipolysis and fatty acid ß-oxidation via increasing energy supply from lipid catabolism, and then, it could economise on the protein from energy production to increase protein deposition in grass carp. Besides, LA might directly promote protein synthesis through activating the mTOR pathway.

Ymr210wp leads to the accumulation of phospholipids and steryl esters in yeast.

Ymr210w was identified as a MAG (Monoacylglycerol) lipase. The accumulation of the phospholipids in the ymr210wΔ was not clearly understood. It was expressed in S. cerevisiae using pYES2/CT vector and His-tag purified recombinant protein confirmed TAG lipase activity. To further evaluate the role of YMR210w, ester hydrolase activity was also confirmed with pNP-acetate, pNP-butyrate and pNP - palmitate. GC-MS lipid profiling of ymr210wΔ showed an increase in the 15:0 Pentadecanoic acid by 76% among the total lipids. Phospholipid, Erucic acid 22:1 (Δ13) showed 43% increase while steryl esters showed significant changes with 16:0 hexadecanoic acid augmentations by 80% and 18:0 Octadecanoic acid by 165% when compared to wild type (WT). Increase in the steryl ester and TAG content supports the accumulation of lipid bodies in ymr210wΔ strain when compared with WT cells.

Comparative analysis of effects of dietary arachidonic acid and EPA on growth, tissue fatty acid composition, antioxidant response and lipid metabolism in juvenile grass carp, Ctenopharyngodon idellus.

Four isonitrogenous and isoenergetic purified diets containing free arachidonic acid (ARA) or EPA (control group), 0·30 % ARA, 0·30 % EPA and 0·30 % ARA+EPA (equivalent) were designed to feed juvenile grass carp (10·21 (sd 0·10) g) for 10 weeks. Only the EPA group presented better growth performance compared with the control group (P<0·05). Dietary ARA and EPA were incorporated into polar lipids more than non-polar lipids in hepatopancreas but not intraperitoneal fat (IPF) tissue. Fish fed ARA and EPA showed an increase of serum superoxide dismutase and catalase activities, and decrease of glutathione peroxidase activity and malondialdehyde contents (P<0·05). The hepatopancreatic TAG levels decreased both in ARA and EPA groups (P<0·05), accompanied by the decrease of lipoprotein lipase (LPL) activity in the ARA group (P<0·05). Fatty acid synthase (FAS), diacylglycerol O-acyltransferase and apoE gene expression in the hepatopancreas decreased in fish fed ARA and EPA, but only the ARA group exhibited increased mRNA level of adipose TAG lipase (ATGL) (P<0·05). Decreased IPF index and adipocyte sizes were found in the ARA group (P<0·05). Meanwhile, the ARA group showed decreased expression levels of adipogenic genes CCAAT enhancer-binding protein α, LPL and FAS, and increased levels of the lipid catabolic genes PPAR α, ATGL, hormone-sensitive lipase and carnitine palmitoyltransferase 1 (CPT-1) in IPF, whereas the EPA group only increased PPAR α and CPT-1 mRNA expression and showed less levels than the ARA group. Overall, dietary EPA is beneficial to the growth performance, whereas ARA is more potent in inducing lipolysis and inhibiting adipogenesis, especially in IPF. Meanwhile, dietary ARA and EPA showed the similar preference in esterification and the improvement in antioxidant response.

Dietary zinc addition influenced zinc and lipid deposition in the fore- and mid-intestine of juvenile yellow catfish Pelteobagrus fulvidraco.

The present study explored the mechanisms of dietary Zn influencing Zn and lipid deposition in the fore- and mid- intestine in yellow catfish Pelteobagrus fulvidraco, and investigated whether the mechanism was intestinal-region dependent. For this purpose, yellow catfish were fed three diets containing Zn levels of 8·83, 19·20 and 146·65 mg Zn/kg, respectively. Growth performance, intestinal TAG and Zn contents as well as activities and mRNA expression of enzymes and genes involved in Zn transport and lipid metabolism in the fore- and mid-intestine were analysed. Dietary Zn increased Zn accumulation as well as activities of Cu-, Zn-superoxide dismutase and ATPase in the fore- and mid-intestine. In the fore-intestine, dietary Zn up-regulated mRNA levels of ZnT1, ZnT5, ZnT7, metallothionein (MT) and metal response element-binding transcription factor-1 (MTF-1), but down-regulated mRNA levels of ZIP4 and ZIP5. In the mid-intestine, dietary Zn up-regulated mRNA levels of ZnT1, ZnT5, ZnT7, MT and MTF-1, but down-regulated mRNA levels of ZIP4 and ZIP5. Dietary Zn reduced TAG content, down-regulated activities of 6-phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME) and fatty acid synthase (FAS) activities, and reduced mRNA levels of 6PGD, G6PD, FAS, PPARγ and sterol-regulator element-binding protein (SREBP-1), but up-regulated mRNA levels of carnitine palmitoyltransferase IA, hormone-sensitive lipase (HSLa), adipose TAG lipase (ATGL) and PPARα in the fore-intestine. In the mid-intestine, dietary Zn reduced TAG content, activities of G6PD, ME, isocitrate dehydrogenase and FAS, down-regulated mRNA levels of 6PGD, G6PD, FAS, acetyl-CoA carboxylase a, PPARγ and SREBP-1, but up-regulated mRNA expression of HSLa, ATGL and PPARγ. The reduction in TAG content following Zn addition was attributable to reduced lipogenesis and increased lipolysis, and similar regulatory mechanisms were observed between the fore- and mid-intestine.

Analysis of the lipid body proteome of the oleaginous alga Lobosphaera incisa.

BACKGROUND: Lobosphaera incisa (L. incisa) is an oleaginous microalga that stores triacylglycerol (TAG) rich in arachidonic acid in lipid bodies (LBs). This organelle is gaining attention in algal research, since evidence is accumulating that proteins attached to its surface fulfill important functions in TAG storage and metabolism. RESULTS: Here, the composition of the LB proteome in L incisa was investigated by comparing different cell fractions in a semiquantitative proteomics approach. After applying stringent filters to the proteomics data in order to remove contaminating proteins from the list of possible LB proteins (LBPs), heterologous expression of candidate proteins in tobacco pollen tubes, allowed us to confirm 3 true LBPs: A member of the algal Major Lipid Droplet Protein family, a small protein of unknown function and a putative lipase. In addition, a TAG lipase that belongs to the SUGAR DEPENDENT 1 family of TAG lipases known from oilseed plants was identified. Its activity was verified by functional complementation of an Arabidopsis thaliana mutant lacking the major seed TAG lipases. CONCLUSIONS: Here we describe 3 LBPs as well as a TAG lipase from the oleaginous microalga L. incisa and discuss their possible involvement in LB metabolism. This study highlights the importance of filtering LB proteome datasets and verifying the subcellular localization one by one, so that contaminating proteins can be recognized as such. Our dataset can serve as a valuable resource in the identification of additional LBPs, shedding more light on the intriguing roles of LBs in microalgae.

Molecular characterization of human ABHD2 as TAG lipase and ester hydrolase.

Alterations in lipid metabolism have been progressively documented as a characteristic property of cancer cells. Though, human ABHD2 gene was found to be highly expressed in breast and lung cancers, its biochemical functionality is yet uncharacterized. In the present study we report, human ABHD2 as triacylglycerol (TAG) lipase along with ester hydrolysing capacity. Sequence analysis of ABHD2 revealed the presence of conserved motifs G(205)XS(207)XG(209) and H(120)XXXXD(125) Phylogenetic analysis showed homology to known lipases, Drosophila melanogaster CG3488. To evaluate the biochemical role, recombinant ABHD2 was expressed in Saccharomyces cerevisiae using pYES2/CT vector and His-tag purified protein showed TAG lipase activity. Ester hydrolase activity was confirmed with pNP acetate, butyrate and palmitate substrates respectively. Further, the ABHD2 homology model was built and the modelled protein was analysed based on the RMSD and root mean square fluctuation (RMSF) of the 100 ns simulation trajectory. Docking the acetate, butyrate and palmitate ligands with the model confirmed covalent binding of ligands with the Ser(207) of the GXSXG motif. The model was validated with a mutant ABHD2 developed with alanine in place of Ser(207) and the docking studies revealed loss of interaction between selected ligands and the mutant protein active site. Based on the above results, human ABHD2 was identified as a novel TAG lipase and ester hydrolase.