Relationship of the Topological Distances and Activities between mPGES-1 and COX-2 versus COX-1: Implications of the Different Post-Translational Endoplasmic Reticulum Organizations of COX-1 and COX-2.

In vascular inflammation, prostaglandin E2 (PGE2) is largely biosynthesized by microsomal PGE2 synthase-1 (mPGES-1), competing with other downstream eicosanoid-synthesizing enzymes, such as PGIS, a synthase of a vascular protector prostacyclin (PGI2), to isomerize the cyclooxygenase (COX)-2-derived prostaglandin H2 (PGH2). In this study, we found that a majority of the product from the cells co-expressing human COX-2, mPGES-1, and PGIS was PGE2. We hypothesize that the molecular and cellular mechanisms are related to the post-translational endoplasmic reticulum (ER) arrangement of those enzymes. A set of fusion enzymes, COX-2-linker [10 amino acids (aa)]-PGIS and COX-2-linker (22 amino acids)-PGIS, were created as "The Bioruler", in which the 10 and 22 amino acids are defined linkers with known helical structures and distances (14.4 and 30.8 Å, respectively). Our experiments have shown that the efficiency of PGI2 biosynthesis was reduced when the separation distance increased from 10 to 22 amino acids. When COX-2-10aa-PGIS (with a 14.4 Å separation) was co-expressed with mPGES-1 on the ER membrane, a major product was PGE2, but not PGI2. However, expression of COX-2-10aa-PGIS and mPGES-1 on a separated ER with a distance of ≫30.8 Å reduced the level of PGE2 production. These data indicated that the mPGES-1 is "complex-likely" colocalized with COX-2 within a distance of 14.4 Å. In addition, the cells co-expressing COX-1-10aa-PGIS and mPGES-1 produced PGI2 mainly, but not PGE2. This indicates that mPGES-1 is expressed much farther from COX-1. These findings have led to proposed models showing the different post-translational ER organization between COX-2 and COX-1 with respect to the topological arrangement of the mPGES-1 during vascular inflammation.

The cellular and molecular defense mechanisms of the Candida yeasts against azole antifungal drugs.

The molecular mechanisms supporting resistance to azole antifungals have attracted a great interest during the last decades because of the emergence of clinical resistance to the treatment of fungal infections. The availability of genome sequencing data, of molecular biology tools, and of a large set of clinical and laboratory azole-resistant strains, made the yeasts Candida the biological material of choice to decipher azole resistance mechanisms. The yeast Candida albicans has several cellular ways to resist to azole drugs: decreased affinity of the target protein Erg11p for the drugs, increased biosynthesis of Erg11p, and efflux of the drugs outside the fungal cells. At the molecular level, two main mechanisms are operating: point mutation in the target gene or in transcriptional activator factors, eventually associated to a loss of heterozygosity, and gene duplication that results from the extraordinary plasticity of the genome. This review proposes to explore the different molecular strategies that are used by Candida yeasts to fight azole antifungals.

Phosphodiesterase 3B is localized in caveolae and smooth ER in mouse hepatocytes and is important in the regulation of glucose and lipid metabolism.

Cyclic nucleotide phosphodiesterases (PDEs) are important regulators of signal transduction processes mediated by cAMP and cGMP. One PDE family member, PDE3B, plays an important role in the regulation of a variety of metabolic processes such as lipolysis and insulin secretion. In this study, the cellular localization and the role of PDE3B in the regulation of triglyceride, cholesterol and glucose metabolism in hepatocytes were investigated. PDE3B was identified in caveolae, specific regions in the plasma membrane, and smooth endoplasmic reticulum. In caveolin-1 knock out mice, which lack caveolae, the amount of PDE3B protein and activity were reduced indicating a role of caveolin-1/caveolae in the stabilization of enzyme protein. Hepatocytes from PDE3B knock out mice displayed increased glucose, triglyceride and cholesterol levels, which was associated with increased expression of gluconeogenic and lipogenic genes/enzymes including, phosphoenolpyruvate carboxykinase, peroxisome proliferator-activated receptor gamma, sterol regulatory element-binding protein 1c and hydroxyl-3-methylglutaryl coenzyme A reductase. In conclusion, hepatocyte PDE3B is localized in caveolae and smooth endoplasmic reticulum and plays important roles in the regulation of glucose, triglyceride and cholesterol metabolism. Dysregulation of PDE3B could have a role in the development of fatty liver, a condition highly relevant in the context of type 2 diabetes.

Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation.

The exocytic pathway provides a physical route through which newly synthesized secretory and membrane proteins are deployed to the eukaryote cell surface. For newly synthesized alpha1-antitrypsin (AAT), the modification of its asparagine-linked oligosaccharides by a slow-acting mannosidase partitions the misfolded monomer into the proteasomal degradation pathway. Herein, we asked whether, and how, modification by endoplasmic reticulum mannosidase I (ERManI) contributes to the preferential selection of the misfolded AAT monomer for proteasomal degradation. Transiently expressed mutant and WT AAT variants underwent rapid destabilization in response to an artificially elevated ERManI concentration in the murine hepatoma cell line, Hepa1a. Based on the mannosidase- and lactacystin-sensitive properties of intracellular turnover, a stochastic model is proposed in which the delayed onset of the glycan modification, relative to the duration of nonnative protein structure, coordinates the preferential degradation of the misfolded monomer and spares the native molecule from destruction. Newly synthesized endogenous transferrin underwent degradation in response to an elevated concentration of ERManI, whereas the nonglycosylated secretory glycoprotein albumin was not affected. Taken together, these findings indicate that efficient conformational maturation might function as the initial quality control standard for a broad population of glycoproteins.

The hepatic glycogenoreticular system.

One of the major liver functions is the ability of hepatocytes to store glucose in the form of glycogen for various purposes. Beside glucose production and secretion, the synthesis of glucuronides and ascorbate has been reported to be dependent on the extent of the glycogen stores and on the rate of glycogenolysis in the liver. It is common that the final steps of these pathways are catalysed by intraluminally orientated enzymes of the endoplasmic reticulum, which are supported by transporters for the permeation of substrates and products. On the basis of the close morphological and functional proximity of glycogen, glycogen-dependent pathways and the (smooth) endoplasmic reticulum we propose to use the term glycogenoreticular system for the description of this export-orientated hepatocyte-specific metabolic unit.

Ultrastructural localization of the NADPH-diaphorase activity in the Leydig cells of aging mice.

Recently, it has been shown that nitric oxide may inhibit the Leydig cell steroidogenesis. The present paper describes, by means of NADPH-diaphorase histochemistry, the ultrastructural localization of the enzyme nitric oxide synthase in the Leydig cells of young adult and aging mice. In the young adult mice, the enzymatic reaction was mainly located in the mitochondria and in some clustered cisternae of the smooth endoplasmic reticulum. The nuclear envelope was faintly labeled. In the aging mice, most Leydig cells showed an enhanced enzymatic reaction. Labeled mitochondria were increased in number, and labeled areas of the smooth endoplasmic reticulum were more numerous and extended. In addition, a strong enzymatic reaction was recognized in the nuclear envelope. We conjecture that the impaired steroidogenesis observed in the testis of aging mammals might, at least in part, depend on the increased nitric oxide production in the Leydig cells.

Regulation of Leydig cell steroidogenic function during aging.

This article summarizes a talk on Leydig cell aging presented at the 1999 Annual Meeting of the Society for the Study of Reproduction. In the Brown Norway rat, serum testosterone levels decrease with aging, accompanied by increases in serum FSH. The capacity of Leydig cells to produce testosterone is higher in young than in old rats. Binding studies with hCG revealed reduced receptor number in old vs. young Leydig cells. In response to incubation with LH, cAMP production was found to be reduced in old vs. young Leydig cells, indicating that signal transduction mechanisms in the old cells are affected by aging. Steroidogenic acute regulatory protein and mRNA levels are reduced in old Leydig cells, suggesting that there may be deficits in the transport of cholesterol to the inner mitochondrial membrane of aged cells. The activity of P450 side-chain cleavage enzyme is reduced in old vs. young cells, as are the activities of each of 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/C17-20 lyase, and 17-ketosteroid reductase. Serum LH levels do not differ between young and old rats, and the administration of LH failed to induce old Leydig cells to produce high (young) testosterone levels, suggesting that the cause of age-related reductions in steroidogenesis is not LH deficits. We hypothesized that reactive oxygen, produced as a by-product of steroidogenesis itself, might be responsible for age-related reductions in testosterone production by the Leydig cells. Consistent with this, long-term suppression of steroidogenesis was found to prevent or delay the reduced steroidogenesis that accompanies Leydig cell aging. A possible explanation of this finding is that long-term suppression of steroidogenesis prevents free radical damage to the cells by suppressing the production of the reactive oxygen species that are a by-product of steroidogenesis itself.

Pharmacological and immunohistochemical characterization of calmodulin-stimulated (Ca(2+)+Mg(2+))-ATPase in cultured porcine aortic endothelial cells.

Plasma membrane (Ca(2+)+Mg(2+))-ATPase and Ca(2+) transport activities, best characterized in human erythrocytes, are stimulated by calmodulin and thought to play a crucial role in the termination of cellular Ca(2+) signaling in all cells. In plasma membranes isolated from cultured porcine aortic endothelial cells, the (Ca(2+)+Mg(2+))-ATPase was not readily measured. This is in part because of an overabundance of nonspecific Ca(2+)- and/or Mg(2+)-activated ecto-5'-nucleotide phosphohydrolases. Moreover, addition of exogenous calmodulin (10(-9) to 10(-6) mol/L) produced no measurable stimulation of ATPase activities, suggesting a permanently activated state or, alternatively, a complete lack thereof. To establish and verify the presence of a calmodulin-regulated (Ca(2+)+Mg(2+))-ATPase activity in these endothelial cells, immunohistochemical localization using a monoclonal mouse anti-(Ca(2+)+Mg(2+))-ATPase antibody (clone 5F10) was applied to intact pig aorta endothelium, cultured endothelial monolayers, and isolated endothelial plasma membrane fractions. This approach clearly demonstrated Ca(2+) pump immunoreactivity in each of these preparations. To confirm functional calmodulin stimulation of the (Ca(2+)+Mg(2+))-ATPase, 10(-5) mol/L calmidazolium (R24571) was added to the isolated plasma membrane preparation, which lowered the (Ca(2+)+Mg(2+))-ATPase activity from 143.0 to 78.15 nmol P(i)/mg protein x min(-1). This calmidazolium-reduced activity could then be stimulated 113.1+/-0.8% in a concentration-dependent manner by the addition of exogenous calmodulin (10(-7) to 2 x 10(-6) mol/L) with an EC(50) of 3.45+/-0.04 x 10(-7) mol/L (n=4). This represents a competitive lowering of the apparent calmodulin affinity by approximately 100 compared with other unopposed calmodulin-stimulated processes. Together, these findings support evidence for the presence of a calmodulin-stimulated plasma membrane (Ca(2+)+Mg(2+))-ATPase activity in cultured porcine aortic endothelial cells.

Characterization and subcellular localization of murine and human magnesium-dependent neutral sphingomyelinase.

Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin, an essential lipid constituent of the plasma membrane, lysosomal membranes, endoplasmic reticulum, and the Golgi membrane stacks of mammalian cells. In this study, we report the biochemical and functional characterization and subcellular localization of magnesium-dependent nSMase1 from overexpressing human embryonic kidney (HEK293) cells. Site-directed mutagenesis of conserved residues probably involved in the enzymatic sphingomyelin cleavage as well as the removal of one or both putative transmembrane domains lead to the complete loss of enzymatic activity of human nSMase1 expressed in HEK293 cells. Polyclonal antibodies raised against recombinant mammalian nSMase1 immunoprecipitated and inactivated the enzyme in membrane extracts of overexpressing HEK293 cells and different murine tissues. Cell fractionation combined with immunoprecipitation studies localized the nSMase1 protein predominantly in the microsomal fraction. The enzyme colocalized with marker proteins of the endoplasmic reticulum and the Golgi apparatus in immunocytochemistry. Anti-nSMase1 antibodies did not affect the nSMase activity in the plasma membrane fraction and membrane extracts from murine brain. Our study leads to the conclusion that nSMase1 is one of at least two mammalian neutral sphingomyelinases with different subcellular localization, tissue specificity, and enzymatic properties.

Interaction of the Unc-51-like kinase and microtubule-associated protein light chain 3 related proteins in the brain: possible role of vesicular transport in axonal elongation.

We identified two mammalian ULK1 (Unc-51-like kinase involved in neurite extension) binding proteins by yeast two-hybrid screening. Both proteins showed high structural similarity to microtubule-associated protein (MAP) light chain 3 (LC3). One is identical to the Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), an essential factor for intra-Golgi transport [39]. The other is identical to the gamma 2-subunit of GABA-A receptor associated protein (GABARAP) which has a possible role in receptor transport [46]. Using the yeast two-hybrid system and the in vitro GST pull-down assay, we found that the N-terminal proline/serine rich (PS) domain of ULK1 (amino acid 287-416) is required for ULK1-GATE-16 and ULK1-GABARAP protein interactions. However, the kinase activity of ULK1 affected neither ULK1-GATE-16 nor ULK1-GABARAP interaction. Immunohistochemical analysis using ULK1 and GABARAP antibodies showed that the ULK1 and the GABARAP proteins co-localized to many kind of neurons such as pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, and Purkinje cells of the cerebellum. In HeLa cells, endogenous ULK1 and tagged GABARAP showed punctate structures in the cytosol, and were colocalized. These results suggest that the interaction of ULK1 and GABARAP is important to vesicle transport and axonal elongation in mammalian neurons.

Autodegradation of protein disulfide isomerase.

Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation.

Adrenergic nerve smooth endoplasmic reticulum calcium buffering declines with age.

Calcium buffering capacity declines with age in sympathetic nerves of rat tail artery. To test whether smooth endoplasmic reticulum (SER) calcium buffering declines with age, effects of two SER calcium-ATPase inhibitors on norepinephrine release and intracellular calcium were determined. Thapsigargin or cyclopiazonic acid caused a significant increase in stimulation-evoked norepinephrine release from 6 month tail arteries with much less effect in 20 months. In isolated superior cervical ganglion cells, the rate of rise of calcium with K+-depolarization increased only in young cells with either cyclopiazonic acid or thapsigargin, with no effect in the old. In young cells, cyclopiazonic acid significantly influenced time to peak, rate of decline, and time to basal of K+-evoked calcium transients, but had no effect in old cells. Thapsigargin caused a significant increase in rate of decline in young, but not old, cells. These differential effects suggest an age-related decline in function of SER calcium buffering mechanisms in the sympathetic nervous system causing older nerves to become more reliant on mitochondria to buffer calcium.

An NADH-dependent disulfide reductase activity in the endoplasmic reticulum of Dictyostelium discoideum.

A novel disulfide reductase activity was observed in the amoeba, Dictyostelium discoideum. Both 5,5'-dithiobis(2-nitro-benzoate) (DTNB) and insulin were substrates for reduction. NADH was utilized in preference to NADPH. The activity comigrated with NADPH-dependent cytochrome c reductase on sucrose gradients, suggesting localization in the endoplasmic reticulum (ER). The buoyant density of the disulfide reductase was not shifted when ribosomes were released from the ER nor was the activity solubilized when membranes were shocked with low osmotic buffer. It therefore appears that the reductase was membrane-bound in the lumen of the smooth ER.

Characterization and development of an inner ear type I fibrocyte cell culture.

A method has been developed that allows successful maintenance of secondary cell cultures derived from explants of the cochlear lateral wall of young adult gerbils. The secondary cultures were characterized morphologically with light and transmission electron microscopy and immunocytochemically with protein markers specific to various lateral wall cell types. Structural studies revealed fusiform-shaped cells with a paucity of cytoplasm surrounding the nucleus and slender processes. The cells showed little evidence of intercellular contact even when confluent. The cultures were immunopositive for vimentin, carbonic anhydrase isozyme II, creatine kinase isozyme BB and smooth endoplasmic reticulum Ca-ATPase, but lacked reactivity for cytokeratins and Na,K-ATPase. The results indicate that the cultures are comprised of type I fibrocytes from the spiral ligament. These findings are the first to demonstrate that inner ear spiral ligament cells can be isolated and maintained in secondary culture while retaining many of their in vivo characteristics. Based upon their location and content of ion transport enzymes, type I fibrocytes are thought to be involved in the recycling of potassium from perilymph into the stria vascularis. The establishment of this cell line provides a means to analyze the role of spiral ligament fibrocytes in maintenance of inner ear homeostasis.

Subcellular localization of the type 2 11beta-hydroxysteroid dehydrogenase. A green fluorescent protein study.

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the inherently non-selective mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. Recently, we characterized a novel isoform of 11beta-HSD in aldosterone target cells, which has high affinity for its substrate, is unidirectional, and prefers NAD as cofactor. In this study we utilized a green fluorescent protein (GFP) technique to determine the subcellular localization of this isoform, 11beta-HSD2. We generated a chimeric gene encoding the full-length rabbit 11beta-HSD2 and, fused to its C terminus, the coding sequence of GFP. This construct was stably transfected into CHO cells. The enzymatic characteristics of the expressed 11beta-HSD2/GFP fusion protein were undistinguishable from those of the native enzyme: high affinity for corticosterone (KM 8-10 nM), NAD dependence, and lack of reductase activity. The intracellular location of the recombinant protein was determined by fluorescence microscopy. 11beta-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm and nuclear envelope, whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Staining of CHO cells expressing 11beta-HSD2/GFP with established subcellular organelle markers revealed a colocalization of 11beta-HSD2/GFP only with ER markers and tubulin. To examine the orientation of 11beta-HSD2 within the ER, we selectively permeabilized CHO cells and stained them with an anti-GFP antibody. Fluorescence microscopy indicated that the C-terminal region of 11beta-HSD2 is on the cytoplasmic surface of the ER membrane, since it was accessible to the GFP antibody. This conclusion was confirmed by trypsin treatment of permeabilized cells followed by Western blotting. The C-terminal region of 11beta-HSD2 was accessible to trypsin, indicating that it is on the cytoplasmic side of the ER membrane. These results indicate that 11beta-HSD2 is localized exclusively to the ER. Since 11beta-HSD2 does not contain any known ER retrieval signal, experiments are currently under way to determine what structural motifs are responsible for its ER localization.

A disulfide-bonded dimer of the Golgi beta-galactoside alpha2,6-sialyltransferase is catalytically inactive yet still retains the ability to bind galactose.

The alpha2,6-sialyltransferase is a terminal glycosyltransferase localized in the trans Golgi and trans Golgi network. Here we show that 30% of the total rat liver Golgi alpha2,6-sialyltransferase forms a disulfide-bonded 100-kDa species that can be converted to the 50-kDa monomer form of the enzyme upon reduction. Limited proteolysis of both enzyme forms demonstrates that the 100-kDa species is a disulfide-bonded homodimer of the alpha2,6-sialyltransferase. The alpha2,6-sialyltransferase disulfide-bonded dimer is found in bovine liver Golgi membranes and in Golgi membranes prepared and solubilized in the presence of 100 mM iodoacetamide, suggesting that it is not unique to rat liver or formed aberrantly upon membrane lysis. The dimer form of the enzyme possesses no significant catalytic activity and has a much lower affinity for CDP-hexanolamine-agarose compared with the monomer form. In contrast, both the alpha2,6-sialyltransferase monomer and the disulfide-bonded dimer bind strongly to galactose and galactose-terminated substrates. These results suggest that the alpha2,6-sialyltransferase disulfide-bonded dimer lacks catalytic activity due to a weak affinity for its sugar nucleotide donor, CMP-NeuAc, and that this catalytically inactive form of the enzyme may act as a galactose-specific lectin in the Golgi.

The association of phosphorylase kinase with membranes of rat liver smooth endoplasmic reticulum.

Upon fractionation of a post mitochondrial supernatant from rat liver, phosphorylase kinase activity was largely recovered in the cytosol and the smooth endoplasmic reticulum (SER) fraction. The presence of phosphorylase kinase in SER vesicles was not due to an interaction of the enzyme with glycogen particles, since previous elimination of SER glycogen either by 48 h animal starvation or by treatment of the membrane fraction with alpha-amylase did not significantly alter phosphorylase kinase activity content. Washing of the initial pellet of SER fraction (crude SER) by dilution and recentrifugation, released in the supernatant an amount of phosphorylase kinase activity, which is dependent on: i) the degree of dilution, ii) the number of washes, iii) the ionic strength of the washing solution and iii) the presence or absence of Ca2+. Crude SER-associated phosphorylase kinase was marginally affected by increased concentrations of antibody against rabbit skeletal muscle holoenzyme which nevertheless drastically inhibited cytosolic enzyme activity, while it showed a higher resistance to partial proteolysis and a different Western blotting profile with anti-phosphorylase kinase when compared with the soluble kinase. A small but significant fraction of SER phosphorylase kinase was strongly associated with the microsomal fraction being partly extractable only in presence of detergents. This membrane-bound enzyme form exhibited an alkaline pH optimum, in contrast to the neutral pH optima of both soluble and weakly associated phosphorylase kinase.

Lead affects steroidogenesis in rat Leydig cells in vivo and in vitro.

Lead is known to impede the male reproductive function, however, the mechanisms through which the adverse effects are mediated are not clearly elucidated. In order to get insight into those mechanisms, we have examined the effects of lead on the biosynthesis of steroid hormones by Leydig cells in the rat. To determine whether lead has a direct action on Leydig cells, we have compared the concentrations of testosterone secreted by Leydig cells in ex vivo experiments after animals had been injected with high doses of lead and in vitro experiments with Leydig cells from normal rats maintained in culture in presence or absence of lead. In ex vivo experiments male Spargue-Dawley rats were injected i.p. with lead acetate (8 mg lead/kg/day, 5 days a week for 5 weeks) or with sodium acetate. Testosterone production by Leydig cells isolated and maintained in culture for 48 h was then assessed under basal conditions or after stimulation by human chorionic gonadotrophin (hCG). Both basal and hCG-stimulated testosterone production dropped by 59% and 37%, respectively, with Leydig cells from lead-exposed rats. For in vitro experiments, cultures of Leydig cells from control rats were exposed to various concentrations of lead acetate for different periods. Dose and time-dependent reductions of testosterone level were observed in the culture medium. The effective doses of hCG for maximal and half-maximal testosterone production did not change, indicating that the sensitivity of Leydig cells to hCG was not impaired by exposure to lead in vitro. Progesterone production was also decreased after this exposure. The negative effect of lead on testosterone and progesterone production was correlated with the lower expression of the enzymes cytochromes P450scc (CYP11A1) and P450c17 (CYP17) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) involved in steroid hormone biosynthesis, as shown by immunohistochemistry. Ultrastructural alterations of the smooth endoplasmic reticulum observed after lead administration might be correlated with the lower expression of the microsomal enzymes P450c17 and 3 beta-HSD. Our results indicate that lead can adversely affect the Leydig cell function by impairing directly steroidogenesis.