Lipid synthesis in cultured human embryonic fibroblasts

We describe here the pathways by which human embryonic fibroblasts synthesize lipids. In these studies, we quantitated the phospholipids by their phosphorus content and by their acyl components. These determinations defined both the chemical composition of the cellular membranes as well as their metabolic turnover. Using radiolabeled precursors, we have shown (a) synthesis of the glycerol moiety via glycolysis and the action of glycerokinase, (b) utilization of both exogenously added and endogenously synthesized fatty acids, (c) synthesis de novo of phosphatidyl choline and phosphatidyl ethanolamine from their base precursors, and (d) the methylation of phosphatidyl ethanolamine yielding phosphatidyl choline. Dividing cells synthesized phosphoglyceride more rapidly than cells in the stationary phase. However, considerable turnover of cellular lipid did occur in the stationary phase.     

An Azide‐Modified Nucleoside for Metabolic Labeling of DNA

Metabolic incorporation of azido nucleoside analogues into living cells can enable sensitive detection of DNA replication through copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) and strain‐promoted azide–alkyne cycloaddition (SPAAC) “click” reactions. One major limitation to this approach is the poor chemical stability of nucleoside derivatives containing an aryl azide group. For example, 5‐azido‐2′‐deoxyuridine (AdU) exhibits a 4 h half‐life in water, and it gives little or no detectable labeling of cellular DNA. In contrast, the benzylic azide 5‐(azidomethyl)‐2′‐deoxyuridine (AmdU) is stable in solution at 37 °C, and it gives robust labeling of cellular DNA upon addition of fluorescent alkyne derivatives. In addition to providing the first examples of metabolic incorporation into and imaging of azide groups in cellular DNA, these results highlight the general importance of assessing azide group stability in bioorthogonal chemical reporter strategies.     

Chloro‐Functionalized Photo‐crosslinking BODIPY for Glutathione Sensing and Subcellular Trafficking

Glutathione (GSH) is one of major antioxidants inside cells that regulates oxidoreduction homeostasis. Recently, there have been extensive efforts to visualize GSH in live cells, but most of the probes available today are simple detection sensors and do not provide details of cellular localization. A new fluorescent probe (pcBD2‐Cl), which is cell permeable and selectively reacts with GSH in situ, has been developed. The in situ GSH‐labeled probe (pcBD2–GSH) exhibited quenches fluorescence, but subsequent binding to cellular abundant glutathione S‐transferase (GST) recovers the fluorescence intensity, which makes it possible to image the GSH–GST complex in live cells. Interactions between probe and GST were confirmed by means of photo‐crosslinking under intact live‐cell conditions. Interestingly, isomers of chloro‐functionalized 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) compounds behaved very distinctively inside the cells. Following co‐staining imaging with MitoTracker and mitochondria fractionation upon lipopolysaccharide‐mediated reactive oxygen species induction experiments showed that pcBD2–GSH accumulated in mitochondria. This is the first example of a live‐cell imaging probe to visualize translocation of GSH from the cytosol to mitochondria.     

Bioorthogonal Probes for Imaging Sterols in Cells

Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C₁₉ alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)‐catalyzed azide–alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two‐color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease.     

The effect of elaidic acid incorporation upon the lipid composition of ehrlich ascites tumor cells and of the host’s liver

The incorporation of elaidic acid into Ehrlich ascites tumor cells (EATC) upon feeding the host an elaidic acid-rich diet has been investigated in the present study. The EATC lipids contained only one-half the concentration of elaidic acid found in the lipids of either the host livers or of livers from normal mice. On the other hand, elaidic acid incorporation into tumor cells was close to that of ascites fluid. This incorporation was mainly into phospholipids; the highest into choline phospholipids and ethanolamine phospholipids. Some changes in the EATC fatty acid composition were noted due to this incorporation. EATC phospholipids had reduced polyunsaturated fatty acids as compared with oleic acid-grown cells. The same was true with respect to ascites fluid phospholipids, but neutral lipids were not altered. Tumor development was accompanied by an increase in elaidic acid of the host’s liver. Elaidic acid incorporation into tumor cells resulted in a reduction in the amount of all major lipids in the tumor. In contrast, elaidic acid had no effect on lipid composition of livers from normal mice and-tumor bearing mice, and also had no effect upon the lipids of the ascites fluid that bathes the tumor cells. The incorporation of elaidic acid into the lipids of EATC, normal liver and host liver did not affect the relative composition of phospholipids in these tissues. The development of the tumor did result in decreases in triacylglycerols and esterified cholesterol, and increases in phospholipids and free cholesterol in the livers of host animals.     

In Vivo Incorporation of Azide Groups into DNA by Using Membrane‐Permeable Nucleotide Triesters

Metabolic incorporation of bioorthogonal functional groups into cellular nucleic acids can be impeded by insufficient phosphorylation of nucleosides. Previous studies found that 5azidomethyl‐2′‐deoxyuridine (AmdU) was incorporated into the DNA of HeLa cells expressing a low‐fidelity thymidine kinase, but not by wild‐type HeLa cells. Here we report that membrane‐permeable phosphotriester derivatives of AmdU can exhibit enhanced incorporation into the DNA of wild‐type cells and animals. AmdU monophosphate derivatives bearing either 5′‐bispivaloyloxymethyl (POM), 5′‐bis‐(4‐acetoxybenzyl) (AB), or “Protide” protective groups were used to mask the phosphate group of AmdU prior to its entry into cells. The POM derivative “POM‐AmdU” exhibited better chemical stability, greater metabolic incorporation efficiency, and lower toxicity than “AB‐AmdU”. Remarkably, the addition of POM‐AmdU to the water of zebrafish larvae enabled the biosynthesis of azide‐modified DNA throughout the body.     

Biosynthesis of phospholipids in subcellular particles from cultured cells of human tissue

A time study of the incorporation of³²P_i into the phospholipids of HeLa, KB, human heart, and liver tissue-culture cell lines has been carried out. The incorporation of³²P_i at various time-intervals into the phospholipids of nuclei, mitochondria, and microsomes of HeLa and KB cells was investigated. The labeling of the isotope into the phospholipids was divided into three groups. The first had two components: phosphatidyl inositol and polyglycerol phosphatides, which showed the greatest incorporation of the isotope as demonstrated in the specific activity values and the percentage of total radioactivity after 15 to 30 minutes of incubation. A second group was composed of the major phospholipids of all tissue-culture cell lines studied, phosphatidyl choline, and phosphatidyl ethanolamine. At first, there was a delayed labeling of these phospholipids; however, after one hour of incubation, a rapid increase was shown in the incorporation of³²P_i. A third group of lipids containing sphingomyelin and phosphatidyl serine demonstrated low specific activity values. The phospholipids of the subcellular fractions, nuclei, mitochondria, and microsomes, had a high degree of incorporation of the isotope into the individual phospholipids and probably represented an active process in the membranes of these cellular units or a renewal of the biological membrane structures. Part of a thesis submitted to the Graduate School of the University of North Dakota in partial fulfillment for the degree of Doctor of Philosophy.     

A Method to Generate and Analyze Modified Myristoylated Proteins

Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motifs, coupled with bio‐orthogonal chemistry, have been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP‐ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP were measured and compared with the unmodified myristoylated Arf1. The ketone‐modified myristoylated Arf1 could be further labeled by fluorophore‐coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid‐modified proteins with additional functions before applying them to cellular systems.     

Ultrastructural Imaging of Salmonella–Host Interactions Using Super‐resolution Correlative Light‐Electron Microscopy of Bioorthogonal Pathogens

The imaging of intracellular pathogens inside host cells is complicated by the low resolution and sensitivity of fluorescence microscopy and by the lack of ultrastructural information to visualize the pathogens. Herein, we present a new method to visualize these pathogens during infection that circumvents these problems: by using a metabolic hijacking approach to bioorthogonally label the intracellular pathogen Salmonella Typhimurium and by using these bioorthogonal groups to introduce fluorophores compatible with stochastic optical reconstruction microscopy (STORM) and placing this in a correlative light electron microscopy (CLEM) workflow, the pathogen can be imaged within its host cell context Typhimurium with a resolution of 20 nm. This STORM‐CLEM approach thus presents a new approach to understand these pathogens during infection.     

Incorporation of fatty acids into phospholipids in L cells stimulated by antibody

Binding antibodies to surface membranes stimulated incorporation of fatty acids (FA) into phospholipids of L cells. Antibodies stimulated at least a 3.4-fold greater incorporation of arachidonic acid into phosphatidylinositol than into any other class of phospholipid when compared on a molar basis (p<0.003). This enhanced incorpoation was selective, depending on the character of the FA, because antibodies stimulated the incorporation of arachidonic acid at least 2.4-fold more than oleic acid, palmitic acid or stearic acid (p<0.001). Surprisingly, an antibody-stimulated incorporation of palmitic acid into sphingomyelin (SM) was at least 2.2-fold greater than that into any other class of phospholipid (p<0.001) and the antibody-stimulated incorporation of palmitic acid into SM was at least 60-fold greater than that of arachidonic acid, stearic or oleic acid (p<0.001). Nontoxic doses of ethylenediamine tetraacetic acid (EDTA), dexamethasone, 4-bromophenacylbromide and indomethacin inhibited the antibody-stimulated incorporation of arachidonic acid into cellular phospholipids, principally phosphatidylinositol (PI), and similarly inhibited the antibody stimulation of DNA synthesis. We conclude that when antibody binds to surface antigens on L cells, a rapid and selective incorporation of fatty acids into certain cellular phospholipids occurs, possibly mediated by calcium-dependent phospholipases. Degradation products of arachidonic acid, i.e., prostaglandins, may be important in these antibody stimulation events, as well. These early changes in phospholipid metabolism may serve as an important signal or mechanism for the subsequent stimulation of DNA synthesis in L cells.     

Selective analysis of lipids by thin-layer chromatography blot matrix-assisted laser desorption/ionization imaging mass spectrometry

Thin-layer chromatography (TLC) is an essential method for food composition analyses such as lipid nutrition analysis. TLC can be used to obtain information about the lipid composition of foods; however, it cannot be used for analyses at the molecular level. Recently we developed a new method that combines matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) with TLC-blotting (TLC-Blot-MALDI-IMS). The combination of MALDI-IMS and TLC blotting enabled detailed and sensitive analyses of lipids. In this study, we applied TLC-Blot-MALDI-IMS for analysis of major phospholipids extracted from bluefin tuna. We showed that TLC-Blot-MALDI-IMS analysis could visualize and identify major phospholipids such as phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin.     

Ubiquitin‐Based Probes Prepared by Total Synthesis To Profile the Activity of Deubiquitinating Enzymes

Epitope‐tagged active‐site‐directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small‐molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor‐intensive intein‐based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active‐site‐directed probes and their application to activity‐based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull‐down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in‐gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull‐down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells.     

Programming in Situ Immunofluorescence Intensities through Interchangeable Reactions of Dynamic DNA Complexes

The regulation of antibody reporting intensities is critical to various in situ fluorescence‐imaging analyses. Although such control is often necessary to visualize sparse molecular targets, the ability to tune marker intensities is also essential for highly multiplexed imaging strategies in which marker reporting levels must be tuned both to optimize dynamic detection ranges and to minimize crosstalk between different signals. Existing chemical amplification approaches generally lack such control. Here, we demonstrate that linear and branched DNA complexes can be designed to function as interchangeable building blocks that can be assembled into organized, fluorescence‐reporting complexes. We show that the ability to program DNA‐strand‐displacement reactions between these complexes offers new opportunities to deterministically tune the number of dyes that are coupled to individual antibodies in order both to increase and controllably balance marker reporting levels within fixed cells.     

Phospholipid synthesis in human embryo fibroblasts infected with herpes simplex virus type 2

The effect of herpes simplex virus type 2 infection on the synthesis of phospholipids in human embryo fibroblasts was determined at temperatures permissive (35 C) or nonpermissive (42 C) for virus replication. Incorporation of [³²P]i was decreased by herpes simplex virus type 2 in fection after 6 hr, which corresponds to the time of initiation of progeny virus production. No differences were observed in the relative incorporation of [³²P]i into phospholipid classes. In another series of experiments, cells were labeled with [³H] ethanolamine before infection and with [¹⁴C] ethanolamine after infection. The incorporation of [¹⁴C] ethanolamine was also decreased after 6 hr of infection. When choline was substituted for ethanolamine, a similar, although less pronounced, decrease in incorporation was seen in infected cells compared to mock-infected cells. During abortive infection at 42 C, incorporation of [³H] thymidine into cellular DNA was stimulated, but the incorporation of phospholipid precursors was decreased. Total phospholipid composition and phospholipid acyl group composition were not changed appreciably during abortive or productive infection, regardless of whether the cells were labeled before or after infection. In conclusion, these data indicated that, during herpes simplex virus type 2 infection, the incorporation of lipid prescursors into phospholipid was decreased. The stimulation of cellular DNA synthesis previously observed during abortive infection at 42 C was not paralleled by a detectable stimulation of total phospholipid synthesis. Neither productive nor abortive infection resulted in significant phospholipid compositional changes in the host cell; however, both resulted in a marked inhibition of phospholipid synthesis.     

Effects of purified eicosapentaenoic acid on arachidonic acid metabolism in cultured murine aortic smooth muscle cells,vessel walls and platelets

The effects of highly purified eicosapentaenoic acid (97% pure) on the arachidonic acid cascade in isolated murine vascular cells and platelets were studied. The incorporation of eicosapentaenoic acid was not as active as that of arachidonic acid in platelets. The ratio of incorporation of eicosapentaenoic acid to arachidonic acid into platelet phospholipids was about 0.7. Analysis of the phospholipid fractions of platelets after labeling with¹⁴C-eicosapentaenoic acid and¹⁴C-arachidonic acid revealed that the incorporation of¹⁴C-eicosapentaenoic acid into the phosphatidylinositol fraction is significantly less than that of¹⁴C-arachidonic acid, while the incorporation of both fatty acids into other phospholipid fractions was almost the same. On the other hand, no significant difference between either fatty acid in incorporation rate, kinetics or distribution in cellular phospholipids was found in cultured aortic smooth muscle cells. Following treatment with eicosapentaenoic acid, cells produced less prostacyclin from endogenous arachidonic acid than did control cells. This was not due to the decrease in fatty acid cyclooxygenase activity, but rather, due to the decrease in arachidonic acid content in cellular phospholipids. In addition, eicosapentaenoic acid was neither converted to prostaglandin I₃ by the vascular cells nor to thromboxane A₃ by platelets. Furthermore, similar results were also obtained by in vivo experiments in which rats were fed with eicosapentaenoic acid enriched diet.     

Identification of Drugs Inducing Phospholipidosis by Novel in vitro Data

Drug‐induced phospholipidosis (PLD) is a lysosomal storage disorder characterized by the accumulation of phospholipids within the lysosome. This adverse drug effect can occur in various tissues and is suspected to impact cellular viability. Therefore, it is important to test chemical compounds for their potential to induce PLD during the drug design process. PLD has been reported to be a side effect of many commonly used drugs, especially those with cationic amphiphilic properties. To predict drug‐induced PLD in silico, we established a high‐throughput cell‐culture‐based method to quantitatively determine the induction of PLD by chemical compounds. Using this assay, we tested 297 drug‐like compounds at two different concentrations (2.5 μM and 5.0 μM ). We were able to identify 28 previously unknown PLD‐inducing agents. Furthermore, our experimental results enabled the development of a binary classification model to predict PLD‐inducing agents based on their molecular properties. This random forest prediction system yields a bootstrapped validated accuracy of 86 %. PLD‐inducing agents overlap with those that target similar biological processes; a high degree of concordance with PLD‐inducing agents was identified for cationic amphiphilic compounds, small molecules that inhibit acid sphingomyelinase, compounds that cross the blood–brain barrier, and compounds that violate Lipinski’s rule of five. Furthermore, we were able to show that PLD‐inducing compounds applied in combination additively induce PLD.     

Relationships between base-exchange reaction and the microsomal phospholipid pool in the rat brain in vitro

The calcium-stimulated incorporation of ethanolamine, choline and L-serine into rat brain microsomal phospholipids has been investigated. The membranes were prelabeled in vitro in their choline or serine phosphoglycerides by base-exchange and then chasing experiments were done by displacing the lipid-bound base by ethanolamine, choline, or L-serine labeled with a different isotope. The results indicate that membrane phosphatidylcholine is presumably a substrate for the exchange with all the three bases, whereas phosphatidylserine exchanges only with ethanolamine and L-serine but not with choline. A small phospholipid pool (3–7% of the total available pool) is active in the calcium-dependent exchange with choline, ethanolamine, and L-serine. When the microsomal membranes are prelabeled in vitro in their phosphatidylcholine moiety through the cytidine-dependent pathway and then chasing experiments are performed with the three nitrogenous bases, as above, the small phospholipid pool is hardly detectable. In view of these and other results (Gaiti et al., FEBS Letters 49:361 1975), it is suggested that at least two different pools of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine might exist in rat brain microsomes.     

A Fluorescent Probe for Imaging Sirtuin Activity in Living Cells,Based on One‐Step Cleavage of the Dabcyl Quencher

Sirtuins (SIRTs) are a family of NAD⁺‐dependent histone deacetylases. In mammals, dysfunction of SIRTs is associated with age‐related metabolic diseases and cancers, so SIRT modulators are considered attractive therapeutic targets. However, current screening methodologies are problematic, and no tools for imaging endogenous SIRT activity in living cells have been available until now. In this work we present a series of simple and highly sensitive new SIRT activity probes. Fluorescence of these probes is activated by SIRT‐mediated hydrolytic release of a 4‐(4‐dimethylaminophenylazo)benzoyl (Dabcyl)‐based FRET quencher moiety from the ?‐amino group of lysine in a nonapeptide derived from histone H3K9 and bearing a C‐terminal fluorophore. The probe SFP3 detected activities of SIRT1, ‐2, ‐3, and ‐6, which exhibit deacylase activities towards long‐chain fatty acyl groups. We then truncated the molecular structure of SFP3 in order to improve both its stability to peptidases and its membrane permeability, and developed probe KST‐F, which showed specificity for SIRT1 over SIRT2 and SIRT3. We show that KST‐F can visualize endogenous SIRT1 activity in living cells.     

Incorporation of acetate into fatty acids and complex lipids of soybean cotyledon slices under aerobic and anaerobic conditions

Soybean slices incubated with [1-¹⁴C] acetate in the presence of air synthesized fatty acids to a greater extent than did slices in the absence of air. The proportion of radioactive fatty acids incorporated into the neutral lipid was ca. 35% in the presence or absence of air. However, both the proportion and the absolute amount of radioactive fatty acids in phospholipids were greater in the presence of air. This difference was particularly great in phosphatidic acid and in a minor uncharacterized phospholipid component. Significant incorporation of acetate into monoenoic acids was observed in these two lipids and in phosphatidyl choline. The latter also showed an accumulation of newly synthesized polyenoic acids when air was present. Stearic acid synthesis was greater under aerobic than under anaerobic conditions. The present results support the concept that a relationship exists between the synthesis of unsaturated fatty acids and their incorporation into phospholipids in plants.