A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

Weill Cornell Medical College

Nucleoside analogues are extensively used in the treatment of cancer and viral diseases. The antiproliferative properties of organorhenium(I) complexes, however, have been scarcely explored to date. Herein we present the syntheses, characterization, and in vitro evaluation of Re(I)(CO)(3) core complexes of thymidine and uridine. For the binding of the Re(I)(CO)(3) core, a tridentate dipicolylamine metal chelate was introduced at positions C5', C2', N3, and C5 with spacers of various lengths. The corresponding organometallic thymidine complexes were fully characterized by IR and NMR spectroscopy and mass spectrometry. Their cytotoxicity was assessed against the A549 lung carcinoma cell line. Toxicity is dependent on the site and mode of conjugation as well as on the nature and the length of the tether. Moderate toxicity was observed for conjugates carrying the rhenium moiety at position C5' or N3 (IC(50)=124-160 microM). No toxicity was observed for complexes modified at C2' or C5. Complex 53, with a dodecylene spacer at C5', exhibits remarkable toxicity and is more potent than cisplatin, with an IC(50) value of 6.0 microM. To the best of our knowledge, this is the first report of the antiproliferative properties of [M(CO)(3)](+1)-nucleoside conjugates. In competitive inhibition experiments with A549 cell lysates and purified recombinant human thymidine kinase 1 (hTK-1), enzyme inhibition was observed for complexes modified at either N3 or C5', but our results suggest that the toxicity cannot be attributed solely to interaction with hTK-1.

Synthesis, cytotoxicity, and insight into the mode of action of Re(CO)3 thymidine complexes.

Sterols are major lipids in eukaryotes and differ in their specific structure between species. Both cholesterol and ergosterol can form liquid ordered domains in artificial membranes. We reasoned that substituting the main sterol ergosterol by cholesterol in yeast should permit domain formation and discriminate between physical and sterol structure-dependent functions. Using a cholesterol-producing yeast strain, we show that solute transporters for tryptophan and arginine are functional, whereas the export of weak organic acids via Pdr12p, a multi-drug resistance family member, is not. The latter reveals a sterol function that is probably dependent upon a precise sterol structure. We present a series of novel yeast strains with different sterol compositions as valuable tools to characterize sterol function and use them to refine the sterol requirements for Pdr12p. These strains will also be improved hosts for heterologous expression of sterol-dependent proteins and safe sources to obtain pure cholesterol and other sterols.

A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance.

Tgl3p, the major triacylglycerol lipase of the yeast Saccharomyces cerevisiae, is a component of lipid droplets but is also present in the endoplasmic reticulum in a minor amount. Recently, it was shown that this enzyme can also serve as a lysophospholipid acyltransferase (Rajakumari, S., and Daum, G. (2010) Mol. Biol. Cell 21, 501-510). Here, we describe the effects of the presence/absence of triacylglycerols and lipid droplets on the functionality of Tgl3p. In a dga1Δlro1Δare1Δare2Δ quadruple mutant lacking all four triacylglycerol- and steryl ester-synthesizing acyltransferases and consequently the lipid droplets, the gene expression of TGL3 was only slightly altered. In contrast, protein level and stability of Tgl3p were markedly reduced in the absence of lipid droplets. Under these conditions, the enzyme was localized to the endoplasmic reticulum. Even the lack of the substrate, triacylglycerol, affected stability and localization of Tgl3p to some extent. Interestingly, Tgl3p present in the endoplasmic reticulum seems to lack lipolytic as well as acyltransferase activity as shown by enzymatic analysis and lipid profiling. Thus, we propose that the activity of Tgl3p is restricted to lipid droplets, whereas the endoplasmic reticulum may serve as a parking lot for this enzyme.

Regulation of the yeast triacylglycerol lipase TGl3p by formation of nonpolar lipids.

Lipid droplets (LD) are in the spotlight of lipid research because of the link of lipid storage to health and disease and the just incipient understanding of their involvement in cellular processes apart from nonpolar lipid metabolism. Yeast is an excellent model organism to study the lipidome and proteome of LD under different environmental conditions and to address new aspects of LD biology and chemistry. In this chapter, we describe a versatile protocol for the isolation of LD at high purity and address specific demands for handling different yeast species. Moreover, we discuss the analysis of the LD proteome and lipidome based on standard methods such as thin layer chromatography (TLC), gas liquid chromatography (GLC), mass spectrometry (MS) as well as GLC/MS. Finally, we point out similarities and disparities of LD proteome and lipidome from the three different yeasts Saccharomyces cerevisiae, Yarrowia lipolytica, and Pichia pastoris.

Analysis of yeast lipid droplet proteome and lipidome.

In the yeast, Saccharomyces cerevisiae, the synthesis of the essential phospholipid phosphatidylethanolamine (PE) is accomplished by a network of reactions which comprises four different pathways. The enzyme contributing most to PE formation is the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) which catalyzes conversion of phosphatidylserine (PS) to PE. To study the genome wide effect of an unbalanced cellular and mitochondrial PE level and in particular the contribution of Psd1p to this depletion we performed a DNA microarray analysis with a ∆psd1 deletion mutant. This approach revealed that 54 yeast genes were significantly up-regulated in the absence of PSD1 compared to wild type. Surprisingly, marked down-regulation of genes was not observed. A number of different cellular processes in different subcellular compartments were affected in a ∆psd1 mutant. Deletion mutants bearing defects in all 54 candidate genes, respectively, were analyzed for their growth phenotype and their phospholipid profile. Only three mutants, namely ∆gpm2, ∆gph1 and ∆rsb1, were affected in one of these parameters. The possible link of these mutations to PE deficiency and PSD1 deletion is discussed.

Transcriptional response to deletion of the phosphatidylserine decarboxylase Psd1p in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317-23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301-37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source.

Screening for hydrolytic enzymes reveals Ayr1p as a novel triacylglycerol lipase in Saccharomyces cerevisiae.

Lipid droplets are specific organelles for the storage of triacylglycerols and steryl esters. They are surrounded by a phospholipid monolayer with a small but specific set of proteins embedded. Assembly and insertion of proteins into this surface membrane is an intriguing question of lipid droplet biology. To address this question we studied the topology of Tgl3p, the major triacylglycerol lipase of the yeast Saccharomyces cerevisiae, on lipid droplets. Employing the method of limited proteolysis of lipid droplet surface proteins, we found that the C terminus of Tgl3p faces the inside of the organelle, whereas the N terminus is exposed at the cytosolic side of lipid droplets. Detailed analysis of the C terminus revealed a stretch of seven amino acids that are critical for protein stability and functionality. The negative charge of two aspartate residues within this stretch is crucial for lipase activity of Tgl3p. A portion of Tgl3p, which is located to the endoplasmic reticulum, exhibits a different topology. In the phospholipid bilayer of the endoplasmic reticulum the C terminus faces the cytosol, which results in instability of the protein. Thus, the topology of Tgl3p is important for its function and strongly dependent on the membrane environment.

Modifications of the C terminus affect functionality and stability of yeast triacylglycerol lipase Tgl3p.

Tgl3p, Tgl4p and Tgl5p are the major triacylglycerol lipases of the yeast Saccharomyces cerevisiae catalyzing degradation of triacylglycerols stored in lipid droplets. Previous results from our laboratory (Athenstaedt and Daum, 2005, J. Biol. Chem. 280, 37301-37309) demonstrated that a yeast strain lacking all three triacylglycerol lipases accumulates not only triacylglycerols at high amount, but also steryl esters. Here we show a metabolic link between synthesis and mobilization of non-polar lipids. In particular, we demonstrate that a block in tri-acylglycerol degradation in a tgl3∆tgl4∆tgl5∆ triple mutant lacking all major triacylglycerol lipases causes marked changes in non-polar lipid synthesis. Under these conditions formation of triacylglycerols is reduced, whereas steryl ester synthesis is enhanced as shown by quantification of non-polar lipids, in vivo labeling of lipids using [(14)C]oleic acid and [(14)C]acetic acid as precursors, and enzyme analyses in vitro. In summary, this study demonstrates that triacylglycerol metabolism and steryl ester metabolism are linked processes. The importance of balanced storage and degradation of these components for lipid homeostasis in the yeast is highlighted.

Defects in triacylglycerol lipolysis affect synthesis of triacylglycerols and steryl esters in the yeast.

Steryl esters and triacylglycerols are the major storage lipids of the yeast Saccharomyces cerevisiae. Steryl esters are formed in the endoplasmic reticulum by the two acyl-CoA:sterol acyltransferases Are1p and Are2p, whereas steryl ester hydrolysis is catalyzed by the three steryl ester hydrolases Yeh1p, Yeh2p and Tgl1p. To shed light on the regulatory link between steryl ester formation and hydrolysis in the maintenance of cellular sterol and free fatty acid levels we employed yeast mutants which lacked the enzymes catalyzing the degradation of steryl esters. These studies revealed feedback regulation of steryl ester formation by steryl ester hydrolysis although in a Δtgl1Δyeh1Δyeh2 triple mutant the gene expression levels of ARE1 and ARE2 as well as protein levels and stability of Are1p and Are2p were not altered. Nevertheless, the capacity of the triple mutant to synthesize steryl esters was significantly reduced as shown by in vitro and in vivo labeling of lipids with [(14)C]oleic acid and [(14)C]acetate. Enzymatic analysis revealed that inhibition of steryl ester formation occurred at the enzyme level. As the amounts and the formation of sterols and fatty acids were also decreased in the triple mutant we concluded that defects in steryl ester hydrolysis also caused feedback inhibition on the formation of sterols and fatty acids which serve as precursors for steryl ester formation. In summary, this study demonstrates a regulatory link within the steryl ester metabolic network which contributes to non-polar lipid homeostasis in yeast cells.

Birgit Ploier

Department of Biochemistry

Weill Cornell Medical College

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Birgit Ploier

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry

Weill Cornell Medical College

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Department of Biochemistry