HIV protease inhibitors protect apolipoprotein B from degradation by the proteasome: a potential mechanism for protease inhibitor-induced hyperlipidemia.

Highly active anti-retroviral therapies, which incorporate HIV protease inhibitors, resolve many AIDS-defining illnesses. However, patients receiving protease inhibitors develop a marked lipodystrophy and hyperlipidemia. Using cultured human and rat hepatoma cells and primary hepatocytes from transgenic mice, we demonstrate that protease inhibitor treatment inhibits proteasomal degradation of nascent apolipoprotein B, the principal protein component of triglyceride and cholesterol-rich plasma lipoproteins. Unexpectedly, protease inhibitors also inhibited the secretion of apolipoprotein B. This was associated with inhibition of cholesteryl-ester synthesis and microsomal triglyceride transfer-protein activity. However, in the presence of oleic acid, which stimulates neutral-lipid biosynthesis, protease-inhibitor treatment increased secretion of apolipoprotein B-lipoproteins above controls. These findings suggest a molecular basis for protease-inhibitor-associated hyperlipidemia, a serious adverse effect of an otherwise efficacious treatment for HIV infection.

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.

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.

Conservation of eukaryotic sterol homeostasis: new insights from studies in budding yeast.

The model eukaryote Saccharomyces cerevisiae (budding yeast) has provided significant insight into sterol homeostasis. The study of sterol metabolism in a genetically amenable model organism such as yeast is likely to have an even greater impact and relevance to human disease with the advent of the complete human genome sequence. In addition to definition of the sterol biosynthetic pathway, almost to completion, the remarkable conservation of other components of sterol homeostasis are described in this review.

Deletion of murine Arv1 results in a lean phenotype with increased energy expenditure.

BACKGROUND: ACAT-related enzyme 2 required for viability 1 (ARV1) is a putative lipid transporter of the endoplasmic reticulum that is conserved across eukaryotic species. The ARV1 protein contains a conserved N-terminal cytosolic zinc ribbon motif known as the ARV1 homology domain, followed by multiple transmembrane regions anchoring it in the ER. Deletion of ARV1 in yeast results in defective sterol trafficking, aberrant lipid synthesis, ER stress, membrane disorganization and hypersensitivity to fatty acids (FAs). We sought to investigate the role of Arv1 in mammalian lipid metabolism. METHODS: Homologous recombination was used to disrupt the Arv1 gene in mice. Animals were examined for alterations in lipid and lipoprotein levels, body weight, body composition, glucose tolerance and energy expenditure. RESULTS: Global loss of Arv1 significantly decreased total cholesterol and high-density lipoprotein cholesterol levels in the plasma. Arv1 knockout mice exhibited a dramatic lean phenotype, with major reductions in white adipose tissue (WAT) mass and body weight on a chow diet. This loss of WAT is accompanied by improved glucose tolerance, higher adiponectin levels, increased energy expenditure and greater rates of whole-body FA oxidation. CONCLUSIONS: This work identifies Arv1 as an important player in mammalian lipid metabolism and whole-body energy homeostasis.

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.

Homoeostatic systems for sterols and other lipids.

Fatty acids and sterols are vital components of all eukaryotic cells. Both are used as building blocks for numerous cellular processes such as membrane biosynthesis or hormone production (sterols). Furthermore, these compounds elicit a variety of effects intracellularly as they can act as signalling molecules and regulate gene expression. The metabolism of fatty acids and sterols represents a very intricate network of pathways that are regulated in a precise manner in order to maintain lipid homoeostasis within a cell. Using the budding yeast Saccharomyces cerevisiae as a model system, we touch upon some of the aspects of achieving and maintaining this lipid homoeostasis.

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.

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.

Human apolipoprotein E mediates processive buoyant lipoprotein formation in insect larvae.

The expression of human apolipoprotein E in tobacco hornworm larvae causes a dramatic change in the buoyant density of the insect's endogenous lipoproteins. Larvae without apoE have lipoproteins that are found exclusively in the high-density range. Baculovirus-mediated apoE expression results in the conversion of approximately one-fourth of the endogenous lipoproteins to low-density species. This density conversion is progressive and parallels a similar change in apoE density distribution. ApoE is secreted from the lipoprotein producing fat body tissue in a lipid-poor form, but readily associates with circulating insect lipoproteins in the hemolymph where the density conversion takes place. Analysis of the buoyant lipoprotein particles indicates that they contain apoE and insect apolipophorins I and II with few or no other proteins present. Immunoprecipitation of apolipophorins I and II results in coprecipitation of apoE. This association is disrupted by detergent, consistent with the three proteins sharing the same lipoprotein particles. The ability of apoE to influence buoyant lipoprotein formation in an invertebrate system leads us to suggest that small apolipoproteins such as apoE may play a role in buoyant lipoprotein production in mammals.

Secretion and lipid association of human apolipoprotein E in Saccharomyces cerevisiae requires a host mutation in sterol esterification and uptake.

We have expressed a cDNA to human apolipoprotein E (apoE) in Saccharomyces cerevisiae. Secretion of apoE was achieved only by the use of a mutant (upc2) strain of yeast with the phenotype of enhanced uptake and intracellular esterification of exogenous cholesterol. Approximately 40 ng/ml apoE was secreted by upc2 mutants in the absence of media cholesterol. ApoE secretion was increased 2-3-fold upon the inclusion of cholesterol in the growth media. This response to exogenous cholesterol was not mediated at the transcriptional level, since apoE mRNA levels were constant under all culture conditions. Concomitant with the increase in secretion following cholesterol uptake by upc2 strains, approximately 5% of secreted apoE was associated with lipid; polar and non-polar lipids were detected in this lipoprotein fraction. Intracellular degradation of apoE in non-secreting strains of yeast was minimized by the presence of null mutations in both vacuolar proteases with non-specific activity (pep4) and a Golgi endopeptidase with specificity for paired basic residues (kex2). The approach of expressing human apolipoproteins in yeast may identify factors that mediate lipoprotein biosynthesis in higher cells. One such factor could be the mammalian equivalent of the gene product of UPC2.

Role of protein processing, intracellular trafficking and endocytosis in production of and immunity to yeast killer toxin.

Yeast strains harboring M1-dsRNA and its packaging virus ScV-L secrete a disulfide-linked, heterodimeric toxin which kills sensitive yeast cells by disrupting plasma membrane function. The mature toxin is derived from a precursor (preprotoxin) which undergoes post-translational processing steps during export via the established yeast secretory pathway. Cleavage by both the KEX1 and KEX2 endopeptidases is required for expression of killing activity. The same 1.0 kb open reading frame on M1-dsRNA directs the expression of immunity to toxin. Differentially processed derivatives of protoxin, as well as protoxin itself, have been proposed to serve as mediators of immunity. To understand the mechanisms by which the killing and immunity phenotypes can be derived from a common precursor, we have: 1) studied cellular processes implicated in expression of the phenotypes; and 2) developed a system to produce mutants defective in immunity, killing, or both. In the first approach, the role played by both endocytosis and vesicular traffiking in expression of killing and immunity was examined. Strains defective in endocytosis (end1, end2) or vacuolar protein localization (vpl3, vpl6) were transformed with a plasmid encoding killer toxin under control of the pho5 promoter. When induced by phosphate starvation, both end mutants and all vpl mutants expressed killing activity. Immunity to exogenous toxin, however, was significantly decreased in strains carrying both vpl mutant alleles and in one of the endocytosis mutants (end1]. This suicidal phenotype (rex for resistance expression) has been described previously in M1-containing strains as a leaky phenocopy.(ABSTRACT TRUNCATED AT 250 WORDS)

The essential function of protein-disulfide isomerase is to unscramble non-native disulfide bonds.

Protein-disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes dithiol oxidation and disulfide bond reduction and isomerization using the active site CGHC. Haploid pdi1 delta Saccharomyces cerevisiae are inviable, but can be complemented with either a wild-type rat PDI gene or a mutant gene coding for CGHS PDI (shufflease). In contrast, pdi1 delta yeast cannot be complemented with a gene coding for SGHC PDI. In vitro, shufflease is an efficient catalyst for the isomerization of existing disulfide bonds but not for dithiol oxidation or disulfide bond reduction. SGHC PDI catalyzes none of these processes. These results indicate that in vivo protein folding pathways contain intermediates with non-native disulfide bonds, and that the essential role of PDI is to unscramble these intermediates.

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.

Hyperlipidemia and inhibitors of HIV protease.

HIV protease inhibitors have been successfully incorporated into therapy for patients with HIV. These otherwise efficacious treatments present with multiple metabolic side-effects and body habitus changes known as the lipodystrophy syndrome. Direct associations of the lipid abnormalities with protease inhibitor use have been described, and ongoing studies are focused on describing mechanisms for future intervention. Mechanisms based on the molecular identity of the protease inhibitor target with human proteins, interference with aspects critical to lipoprotein production, and interference with adipocyte differentiation have been described. This review highlights the complexities of this syndrome, and discusses putative mechanisms whereby protease inhibitors cause hyperlipidemia.

Determination of pig apolipoprotein B genotype by gene amplification and restriction fragment length polymorphism analysis.

Sequence differences within the pig apoB gene can be used to identify rapidly four of eight known pig apoB alleles, designated LPB1-LBP8. We describe the use of gene amplification, followed by endonuclease digestion and agarose gel electrophoresis, to discern size and restriction site differences. LPB5, a common allele associated with reduced low density lipoprotein clearance and hypercholesterolaemia in pigs, is identified by a 283-bp insertion in intron 28. LPB3 and LPB7 are distinguished by a unique HindIII site; LPB8 shares a unique HincII site with LPB5. This method facilitates identification of the apoB genotype of pigs used in lipoprotein research and allows for further investigation into the association of particular apoB alleles with lipoprotein metabolism abnormalities.

Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin.

Strains of Saccharomyces cerevisiae harboring M1-dsRNA, the determinant of type 1 killer and immunity phenotypes, secrete a dimeric 19-kd toxin that kills sensitive yeast cells by the production of cation-permeable pores in the cytoplasmic membrane. The preprotoxin, an intracellular precursor to toxin, has the domain sequence delta-alpha-gamma-beta where alpha and beta are the 9.5-and 9.0-kd subunits of secreted toxin. Plasmids containing a partial cDNA copy of M1, in which alpha, gamma, and beta are fused to the PH05 promoter and signal peptide, have previously been shown to express phosphate-repressible toxin production and immunity. Here the construction of a complete DNA copy of the preprotoxin gene and its mutagenesis are described. Analysis of the expression of these mutants from the PH05 promoter elucidates the functions of the preprotoxin domains. delta acts as a leader peptide and efficiently mediates the secretion, glycosylation and maturation of killer toxin. Mutations within the beta subunit indicate it to be essential for binding of toxin to and killing of whole cells but unnecessary for the killing of spheroplasts. Mutations within the putative active site of alpha prevent killing of both cells and spheroplasts. The probable role of beta is therefore recognition and binding to the cell wall receptor whereas alpha is the active ionophore. Mutations within alpha causing loss of toxicity also cause loss of immunity, while the mutants described within gamma and beta retain partial or complete immunity. Expression of gamma without alpha or beta confers no phenotype. The immunity determinant may minimally consist of the alpha domain and the N-terminal portion of gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.