Biomedical subjects

S L Sturley

Publications and source records attributed to S L Sturley.

At least 19 recordsLinked to original sources

PubMed2000

A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast.

The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases. Four of these genes are similar to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1 yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor. The residual triglyceride biosynthesis that persists in the LRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol acyltransferase. These mechanisms may also exist in mammalian cells.

Acyltransferases↗

PubMed2000

Endogenously expressed apolipoprotein E has different effects on cell lipid metabolism as compared to exogenous apolipoprotein E carried on triglyceride-rich particles.

Apolipoprotein E (apoE) on model triglyceride-rich particles (TGRP) increases triglyceride (TG) utilization and cholesteryl ester (CE) hydrolysis, independent of its effect on enhancing particle uptake. We questioned whether, under physiological concentrations, endogenously expressed apoE has similar effects on cellular lipid metabolism as compared to exogenous apoE. J774 macrophages, which do not express apoE, were engineered to express endogenous apoE by transfection of human apoE3 cDNA expression constructs (E(+)) or control vectors (E(-)) into the cells. To compare the effects of exogenous apoE and endogenous apoE on TGRP uptake, cells were incubated with or without apoE associated with (3)H-cholesteryl ether-labeled TGRP. Exogenous apoE enhanced TGRP uptake in both E(-) and E(+) cells. E(-) cells displayed significantly higher TGRP uptake than E(+) cells. Sodium chlorate, which inhibits cell proteoglycan synthesis, markedly diminished differences in TGRP uptake between E(-) and E(+) cells, suggesting that endogenous apoE-proteoglycan interaction contributes to differences in uptake between the two cell types. Particle uptake by the LDL receptor, by the LDL receptor related protein, or by scavenger receptors were similar between E(-) and E(+) cells indicating that endogenous apoE expression does not have a general effect on endocytic pathways. Exogenous apoE carried on TGRP stimulated TG utilization and CE hydrolysis in both cell types. However, TG utilization and CE hydrolysis were not affected by endogenous apoE expression. In conclusion, macrophage expression of apoE has very different effects on TGRP metabolism than exogenously supplied apoE. The fluorescence microscopy results in this study showing that exogenous apoE and endogenous apoE were confined in separate cellular compartments support the hypothesis that these differences resulted from distinct intracellular trafficking pathways followed by exogenous apoE bound to TGRP as compared to endogenous cell-expressed apoE.

Apolipoproteins E↗

PubMed2000

Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase.

During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants.

Acyl Coenzyme A↗

PubMed1999

A molecular target for viral killer toxin: TOK1 potassium channels.

Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells. The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death. Here, the toxin is shown to activate the plasma membrane potassium channel of S. cerevisiae, TOK1. Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility. Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not. K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X. laevis oocytes.

Animals↗

PubMed1998

Translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apolipoprotein B are determined by the presence of beta sheet domains.

Apolipoprotein (apo) B100 is an atypical secretory protein in that its translocation across the endoplasmic reticulum membrane is inefficient, resulting in the partial translocation and exposure of apoB100 on the cytoplasmic surface of the endoplasmic reticulum. Cytosolic exposure leads to the association of nascent apoB with heat shock protein 70 and to its predisposition to ubiquitination and proteasomal degradation. The basis for the inefficient translocation of apoB100 remains unclear and controversial. To test the hypothesis that beta sheet domains present in apoB100 contribute to its inefficient translocation, we created human apoB chimeric constructs apoB13,16 and apoB13,13,16, which contain amino-terminal alpha globular domains but no beta sheet domains, and apoB13,16,beta, which has an amphipathic beta sheet domain of apoB100 inserted into apoB13,16. These constructs, along with carboxyl-terminal truncations of apoB100, apoB34 and apoB42, were used to transfect HepG2 and Chinese hamster ovary cells. In contrast to the lack of effect of proteinase K on apoB13,16 and apoB13,13,16, the levels of apoB34, apoB42, and apoB13,16,beta were decreased by 70-85% after proteinase K-induced proteolysis in both HepG2 and Chinese hamster ovary cells. Either oleic acid or proteasomal inhibitors (N-acetyl-leucinyl-leucinyl-norleucinal and lactacystin) significantly increased the cell levels of apoB13,16,beta, apoB34, apoB42, and full-length apoB100 but had no effect on the cell levels of apoB13,16 and apoB13,13,16. When HepG2 cells were incubated with a microsomal triglyceride transfer protein inhibitor, the cellular levels of apoB13,16,beta, apoB34, and apoB42 were decreased by 70-80%, whereas the levels of apoB13,16 and apoB13,13,16 were unaffected. The effects of microsomal triglyceride transfer protein inhibition were reversed by lactacystin. Our results clearly demonstrate that the translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apoB were determined by the presence of a lipid binding beta sheet domain. It is possible that beta sheet domains may at least transiently facilitate the interaction of apoB with the lipid bilayer surrounding the translocation channel.

Apolipoprotein B-100↗

PubMed1998

Characterization of two human genes encoding acyl coenzyme A:cholesterol acyltransferase-related enzymes.

The enzyme acyl coenzyme A:cholesterol acyltransferase 1 (ACAT1) mediates sterol esterification, a crucial component of intracellular lipid homeostasis. Two enzymes catalyze this activity in Saccharomyces cerevisiae (yeast), and several lines of evidence suggest multigene families may also exist in mammals. Using the human ACAT1 sequence to screen data bases of expressed sequence tags, we identified two novel and distinct partial human cDNAs. Full-length cDNA clones for these ACAT related gene products (ARGP) 1 and 2 were isolated from a hepatocyte (HepG2) cDNA library. ARGP1 was expressed in numerous human adult tissues and tissue culture cell lines, whereas expression of ARGP2 was more restricted. In vitro microsomal assays in a yeast strain deleted for both esterification genes and completely deficient in sterol esterification indicated that ARGP2 esterified cholesterol while ARGP1 did not. In contrast to ACAT1 and similar to liver esterification, the activity of ARGP2 was relatively resistant to a histidine active site modifier. ARGP2 is therefore a tissue-specific sterol esterification enzyme which we thus designated ACAT2. We speculate that ARGP1 participates in the coenzyme A-dependent acylation of substrate(s) other than cholesterol. Consistent with this hypothesis, ARGP1, unlike any other member of this multigene family, possesses a predicted diacylglycerol binding motif suggesting that it may perform the last acylation in triglyceride biosynthesis.

Acylation↗

PubMed1998

Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein.

Membrane physiology, plasma lipid levels, and intracellular sterol homeostasis are regulated by both fatty acids and cholesterol. Sterols regulate gene expression of key enzymes of cholesterol and fatty acid metabolism through proteolysis of the sterol regulatory element-binding protein (SREBP), which binds to sterol regulatory elements (SRE) contained in promoters of these genes. We investigated the effect of fatty acids on SRE-dependent gene expression and SREBP. Consistent results were obtained in three different cell lines (HepG2, Chinese hamster ovary, and CV-1) transfected with SRE-containing promoters linked to the luciferase expression vector. We show that micromolar concentrations of oleate and other polyunsaturated fatty acids (C18:2-C22:6) dose-dependently (0.075-0.6 mmol) decreased transcription of SRE-regulated genes by 20-75%. Few or no effects were seen with saturated free fatty acids. Fatty acid effects on SRE-dependent gene expression were independent and additive to those of exogenous sterols. Oleate decreased levels of the mature sterol regulatory element-binding proteins SREBP-1 and -2 and HMG-CoA synthase mRNA. Oleate had no effect in sterol regulation defective Chinese hamster ovary cells or in cells transfected with mutant SRE-containing promoters. We hypothesize that unsaturated fatty acids increase intracellular regulatory pools of cholesterol and thus affect mature SREBP levels and expression of SRE-dependent genes.

Animals↗

PubMed1998

A molecular approach to understanding human sterol metabolism using yeast genetics.

The availability of the sequenced genome of Saccharomyces cerevisiae (yeast) has culminated in the use of this model eukaryote to study human diseases at a basic level. This article describes the advantages of studying lipid metabolism in this genetically facile organism, including examples of conserved functions and genetic approaches to identifying new components of cholesterol homeostasis.

Genome, Fungal↗

PubMed1997

Heparan sulfate proteoglycan-mediated uptake of apolipoprotein E-triglyceride-rich lipoprotein particles: a major pathway at physiological particle concentrations.

We explored potential mechanisms of non-low-density lipoprotein (LDL) receptor-mediated uptake of triglyceride-rich particles (TGRP) in the presence of apolipoprotein E (apo E). Human fibroblasts were incubated with model intermediate-density lipoprotein- (IDL-) sized TGRP (10-1000 microg of neutral lipid/mL) containing apo E. The extent of receptor-mediated uptake of TGRP was assessed with (a) an anti-apo E monoclonal antibody, which blocks receptor interaction; (b) incubation with heparin; (c) normal vs LDL receptor-negative fibroblasts; and (d) receptor-associated protein (RAP) to determine the potential contribution of LDL receptor-related protein (LRP). Cell surface heparan sulfate proteoglycan- (HSPG-) mediated uptake was examined with or without the addition of heparinase and heparitinase to cell incubation mixtures. At low particle concentrations (</=100 microg of neutral lipid/mL), almost all apo E-TGRP uptake was via the LDL receptor. At higher particle concentrations, within the physiologic range (>250 microg of neutral lipid/mL), most (>/=60%) particle uptake and internalization was via HSPG-mediated pathways. This HSPG pathway did not involve classical lipoprotein receptors, such as LRP or the LDL receptor. These data suggest that in peripheral tissues, such as the arterial wall, apo E may act in TGRP as a ligand for uptake not only via the LDL receptor and LRP pathways but also via HSPG pathways that are receptor-independent. Thus, at physiologic particle concentrations apo E-TGRP can be bound and internalized in certain cells by relatively low affinity but high capacity HSPG-mediated pathways.

Antibodies, Monoclonal↗

PubMed1997

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Amino Acid Sequence↗

PubMed1997

Functional expression of a cDNA to human acyl-coenzyme A:cholesterol acyltransferase in yeast. Species-dependent substrate specificity and inhibitor sensitivity.

We have identified two yeast genes with similarity to a human cDNA encoding acyl-coenzyme A:cholesterol acyltransferase (ACAT). Deletion of both yeast genes results in a viable cell with undetectable esterified sterol (Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R. J., Aljinovic, G., Pohl, T., Rothstein, R., and Sturley, S. L. (1996) Science 272, 1353-1356). Here, we expressed the human cDNA in the yeast double mutant, resulting in high level production of ACAT protein, but low in vivo esterification of ergosterol, the predominant yeast sterol. The activity of the human enzyme was increased by incubation of these cells with 25-hydroxy, cholesterol, an established positive regulator of mammalian sterol esterification. In contrast, the yeast enzymes were unaffected by this reagent. In vitro microsomal assays indicated no sterol esterification in extracts from the double mutant. However, significant activity was detected from strains expressing human ACAT when cholesterol was equilibrated with the microsomal membranes. The human enzyme in yeast utilized cholesterol as the preferred sterol and was sensitive to competitive (S58035) and non-competitive (DuP 128) ACAT inhibitors. The yeast esterifying enzymes exhibited a diminished sterol substrate preference and were sensitive only to S58035. Human ACAT had a broad acyl-CoA substrate specificity, the other substrate for this reaction. By contrast, the yeast enzymes had a marked preference for specific acyl-CoAs, particularly unsaturated C18 forms. These results confirm the yeast genes as functional homologs of the human gene and demonstrate that the enzymes confer substrate specificity to the esterification reaction in both organisms.

Cholesterol↗

PubMed1997

Expression and purification of soluble, active heterodimeric guanylyl cyclase from baculovirus.

A method for expression and purification of active cytosolic heterodimeric histidine (His)-tagged guanylyl cyclase of the alpha 1/beta 1 isoform has been developed using recombinant baculovirus-transfected insect cells. Confirmation of expression of active cyclase was obtained by both Western analysis and enzymatic activity. A His tag on the COOH-terminus of the alpha 1 and beta 1 subunits allowed rapid purification of the heterodimeric form of guanylyl cyclase in a single affinity step using a nickel column. A second gel-filtration step was applied to reconstitute into the complex heme, a required cofactor. This was confirmed spectroscopically by absorbance in the Soret region. Like enzyme purified from tissue, the activity of recombinant guanylyl cyclase was increased by protoporphyrin IX and inhibited by both Zn- and Sn-protoporphyrin. The method described here should provide a general approach for the expression and purification of alternate forms of cytosolic guanylyl cyclase and facilitate mechanistic and structural studies of this important family of enzymes. Furthermore, the procedure demonstrates the utility of the His-tag system to purify multimeric proteins.

Animals↗

PubMed1997

Molecular aspects of intracellular sterol esterification: the acyl coenzyme A: cholesterol acyltransferase reaction.

The conversion of cholesterol to an esterified storage form by the enzyme acyl-coenzyme A: cholesterol acyltransferase, is a critical component of sterol and membrane homeostasis and represents a significant step in atherogenesis. The isolation in 1993 of the first molecular probe of acyl-coenzyme A: cholesterol acyltransferase provided a key to understanding this reaction and its regulation. The sequence is apparently ubiquitous in eukaryotes, often with multiple sequence homologs within an organism. Presented here is a review of the known molecular events that lead to intracellular sterol esterification.

Animals↗

PubMed1997

Protein disulfide isomerase in spore germination and cell division.

Protein disulfide isomerase (PDI) is a protein of the endoplasmic reticulum (ER) that is essential for the unscrambling of nonnative disulfide bonds. Here, we have determined the importance of PDI to both spore germination and vegetative cell division. To vary the concentration of PDI in the ER, we used plasmids that direct the expression of rat PDI fused at its N terminus to either the alpha-factor pre-pro segment or the alpha-factor pre sequence, and fused at its C terminus to either the mammalian (KDEL) or the yeast (HDEL) ER retention signal. Classical yeast genetic (tetrad) analyses, and plasmid loss and plasmid shuffling experiments were used to evaluate the ability of these constructs to complement haploid Saccharomyces cerevisiae cells in which the endogenous PDI1 gene had been deleted. We find that basal levels of PDI in the ER are sufficient for vegetative growth. In contrast, high levels of PDI in the ER are required for efficient spore germination. Thus, catalysis of the unscrambling of nonnative disulfide bonds in cellular proteins is more important during spore germination than during vegetative cell division.

Animals↗

PubMed1996

Positive and negative regulation of a sterol biosynthetic gene (ERG3) in the post-squalene portion of the yeast ergosterol pathway.

Regulation of sterol biosynthesis in the terminal portion of the pathway represents an efficient mechanism by which the cell can control the production of sterol without disturbing the production of other essential mevalonate pathway products. We demonstrate that mutations affecting early and late steps in sterol homeostasis modulate the expression of the ERG3 gene: a late step in sterol biosynthesis in yeast. Expression of ERG3 is increased in response to a mutation in the major isoform of HMG CoA reductase which catalyzes the rate-limiting step of sterol biosynthesis. Likewise, mutations in non-auxotrophic ergosterol biosynthetic genes downstream of squalene production (erg2, erg3, erg4, erg5, and erg6) result in an up-regulation of ERG3 expression. Deletion analysis of the ERG3 promoter identified two upstream activation sequences: UAS1 which when deleted reduces ERG3 gene expression 3-4-fold but maintains sterol regulation and UAS2, which when deleted further reduces ERG3 expression and abolishes sterol regulation. The recent isolation of two yeast genes responsible for the esterification of intracellular sterol (ARE1 and ARE2) has enabled us to directly analyze the relationship between sterol esterification and de novo biosynthesis. Our results demonstrate that the absence of sterol esterification leads to a decrease in total intracellular sterol and ERG3 is a target of this negative regulation.

Acyltransferases↗

PubMed1996

Sterol esterification in yeast: a two-gene process.

Unesterified sterol modulates the function of eukaryotic membranes. In human cells, sterol is esterified to a storage form by acyl-coenzyme A (CoA): cholesterol acyl transferase (ACAT). Here, two genes are identified, ARE1 and ARE2, that encode ACAT-related enzymes in yeast. The yeast enzymes are 49 percent identical to each other and exhibit 23 percent identity to human ACAT. Deletion of ARE2 reduced sterol ester levels to approximately 25 percent of normal levels, whereas disruption of ARE1 did not affect sterol ester biosynthesis. Deletion of both genes resulted in a viable cell with undetectable esterified sterol. Measurements of [14C]acetate incorporation into saponified lipids indicated down-regulation of sterol biosynthesis in the are1 are2 mutant cells. With the use of a consensus sequence to the yeast and human genes, an additional number of the ACAT gene family was identified in humans.

Acetates↗

PubMed1996

The amino terminus of apolipoprotein B is necessary but not sufficient for microsomal triglyceride transfer protein responsiveness.

Human apolipoprotein (apo) B mediates the formation of neutral lipid-containing lipoproteins in the liver and intestine. The association of apoB with lipid is thought to be promoted by the microsomal triglyceride transfer protein complex. We have reconstituted lipoprotein assembly in an insect cell line that normally does not support this process. Expression of human microsomal triglyceride transfer protein (MTP) and apolipoprotein B48 (apoB48) together enabled Sf-21 insect cells to secrete approximately 60-fold more lipoprotein-associated triacylglycerol than control cells. This dramatic effect demonstrates that effective partitioning of triacylglycerol into the secretory pathway requires an endoplasmic reticulum-associated neutral lipid transporter (provided by MTP) and an apolipoprotein to shuttle the lipid through the pathway. Expression of the human apoB48 gene in insect cells resulted in secretion of the protein product. Including both MTP subunits with apoB48 and oleic acid specifically increased apoB48 secretion 8-fold over individual subunits alone. To assess whether specific regions of apoB are necessary for MTP responsiveness, nine apoB segments were expressed. These included NH2-terminal segments as well as internal and COOH-terminal regions of apoB fused with a heterologous signal sequence. ApoB segments containing the NH2-terminal 17% of the protein were secreted and responded to MTP activity; however, a segment containing only the NH2-terminal 17% of the protein was not significantly responsive to MTP. Segments lacking the NH2 terminus were not MTP-responsive, and five of six of these proteins were trapped intracellularly but, in certain cases, could be rescued by fusion to apoB17. These results suggest that the NH2 terminus of apoB is necessary but not sufficient for MTP responsiveness.

Animals↗

PubMed1996

Expression and characterization of a truncated, soluble, low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor mediates the clearance of apolipoprotein B- and E-containing lipoproteins from the bloodstream. In the current study, we characterized the binding properties of the amino terminus of the LDL receptor. We produced a recombinant baculovirus that encoded the first 354 amino acids, including the endogenous signal sequence, of the human LDL receptor. This truncated receptor protein (LDL-R354) was secreted from recombinant baculovirus-infected Spodoptera frugiperda (Sf-21) cells. Upon electrophoresis, LDL-R354 migrated with a mobility of 55,000. Treatment of cells with tunicamycin decreased the size of the truncated receptor, suggesting the presence of asparagine-linked carbohydrates. Nonreducing SDS-PAGE resulted in at least three discernible bands with M(r)s consistent with the truncated receptor existing as monomers and multimers, suggesting the possibility of intermolecular disulfide cross-linking. All forms of the truncated receptor bound LDL on a ligand blot in a calcium-dependent manner. The purified truncated receptor bound 125I-LDL with high affinity in a competitive binding assay (IC50 = 0.8 microgram/ml). LDL-R354 also bound calcium. This interaction was sensitive to the conformation of the ligand binding domain, as reduction of the disulfide bonds greatly decreased the affinity of the receptor for calcium. The ligand and calcium binding activities of this truncated receptor protein demonstrate that the ligand binding domain of the LDL receptor can fold into a functionally active protein.

Animals↗