Trehalose renders the dauer larva of Caenorhabditis elegans resistant to extreme desiccation

Water is essential for life on Earth. In its absence, however, some organisms can interrupt their life cycle and temporarily enter an ametabolic state, known as anhydrobiosis [1]. It is assumed that sugars (in particular trehalose) are instrumental for survival under anhydrobiotic conditions [2]. However, the role of trehalose remained obscure because the corresponding evidence was purely correlative and based mostly on in vitro studies without any genetic manipulations of trehalose metabolism. In this study, we used C. elegans as a genetic model to investigate molecular mechanisms of anhydrobiosis. We show that the C. elegans dauer larva is a true anhydrobiote: under defined conditions it can survive even after losing 98% of its body water. This ability is correlated with a several fold increase in the amount of trehalose. Mutants unable to synthesize trehalose cannot survive even mild dehydration. Light and electron microscopy indicate that one of the major functions of trehalose is the preservation of membrane organization. Fourier-transform infrared spectroscopy of whole worms suggests that this is achieved by preserving homogeneous and compact packing of lipid acyl chains. By means of infrared spectroscopy, we can now distinguish a "dry, yet alive" larva from a "dry and dead" one.     

Exogenous Administration of Gangliosides Displaces GPI-anchored Proteins from Lipid Microdomains in Living Cells

Exogenous application of gangliosides to cells affects many cellular functions. We asked whether these effects could be attributed to the influence of gangliosides on the properties of sphingolipid-cholesterol microdomains on the plasma membrane, also termed rafts. The latter are envisaged as lateral assemblies of sphingolipids (including gangliosides), cholesterol, and a specific set of proteins. Rafts have been implicated in processes such as membrane trafficking, signal transduction, and cell adhesion. Recently, using a chemical cross-linking approach with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) as a model system, we could show that GPI-anchored proteins are clustered in rafts in living cells. Moreover, this clustering was dependent on the level of cholesterol in the cell. Here we show that incubation of MDCK cells with gangliosides abolished subsequent chemical cross-linking of GH-DAF. Furthermore, insertion of gangliosides into the plasma membrane of MDCK GH-DAF cells renders GH-DAF soluble when subjected to extraction with Triton X-114 at 4 degrees C. Our data suggest that exogenous application of gangliosides displaces GPI-anchored proteins from sphingolipid-cholesterol microdomains in living cells.     

Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.     

tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products

We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.     

GTP interacts with the gamma-subunit of eukaryotic initiation factor eIF-2

Eukaryotic initiation factor eIF-2 is an oligomeric protein consisting of three different subunits. During initiation of protein synthesis eIF-2 interacts with GTP, Met-tRNAf and 40 S ribosomal subunit. By affinity labeling with a photo-reactive GTP analogue it was shown that in the binary complex [eIF-2 X GTP] GTP is in contact with the gamma-subunit of eIF-2.     

LET-767 is required for the production of branched chain and long chain fatty acids in Caenorhabditis elegans

LET-767 from Caenorhabditis elegans belongs to a family of short chain dehydrogenases/reductases and is homologous to 17beta-hydroxysterol dehydrogenases of type 3 and 3-ketoacyl-CoA reductases. Worms subjected to RNA interference (RNAi) of let-767 displayed multiple growth and developmental defects in the first generation and arrested in the second generation as L1 larvae. To determine the function of LET-767 in vivo, we exploited a biochemical complementation approach, in which let-767 (RNAi)-arrested larvae were rescued by feeding with compounds isolated from wild type worms. The arrest was only rescued by the addition of triacylglycerides extracted from worms but not from various natural sources, such as animal fats and plant oils. The mass spectrometric analyses showed alterations in the fatty acid content of triacylglycerides. Essential for the rescue were odd-numbered fatty acids with monomethyl branched chains. The rescue was improved when worms were additionally supplemented with long chain even-numbered fatty acids. Remarkably, let-767 completely rescued the yeast 3-ketoacyl-CoA reductase mutant (ybr159Delta). Because worm ceramides exclusively contain a monomethyl branched chain sphingoid base, we also investigated ceramides in let-767 (RNAi). Indeed, the amount of ceramides was greatly reduced, and unusual sphingoid bases were observed. Taken together, we conclude that LET-767 is a major 3-ketoacyl-CoA reductase in C. elegans required for the bulk production of monomethyl branched and long chain fatty acids, and the developmental arrest in let-767 (RNAi) worms is caused by the deficiency of the former.     

Caveolin-1 deficiency alters plasma lipid and lipoprotein profiles in mice

Caveolae are specialized membrane microdomains formed as the result of local accumulation of cholesterol, glycosphingolipids, and the structural protein caveolin-1 (Cav-1). To further elucidate the role of Cav-1 in lipid homeostasis in-vivo, we analyzed fasting and post-prandial plasma from Cav-1 deficient mice on low or on high fat diet. In total plasma analysis, an increase in ceramide and hexosylceramide was observed. In cholesteryl ester (CE), we found an increased saturated + monounsaturated/polyunsaturated fatty acid ratio in fasting plasma of low fat fed Cav-1(−/−) mice with increased proportions of CE16:1, CE18:1, CE20:3, and decreased proportions of CE18:2 and CE22:6. Under high fat diet HDL-CE, free cholesterol and pre-β-HDL were increased accompanied by a shift from slow to fast migrating α-HDL and expansion of apoE containing HDL. Our results demonstrate a significant role of Cav-1 in HDL-cholesterol metabolism and may reflect a variety of Cav-1 functions including modulation of ACAT activity and SR-BI function.     

Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics

Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the "gold-standard" Folch or Bligh and Dyer recipes.