Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae

Most Ras proteins are posttranslationally modified by a palmitoyl lipid moiety through a thioester linkage. However, the mechanism by which this occurs is not known. Here, evidence is presented that the Ras2 protein of Saccharomyces cerevisiae is palmitoylated by a Ras protein acyltransferase (Ras PAT) encoded by the ERF2 and ERF4 genes. Erf2p is a 41-kDa protein localized to the membrane of the endoplasmic reticulum and contains a conserved DHHC cysteine-rich domain (DHHC-CRD). Erf2p co-purifies with Erf4p (26 kDa) when it is expressed in yeast or in Escherichia coli. The Erf2p/Erf4p complex is required for Ras PAT activity, and mutations within conserved residues (Cys(189), His(201), and Cys(203)) of the Erf2p DHHC-CRD domain abolish Ras PAT activity. Furthermore, a palmitoyl-Erf2p intermediate is detected suggesting that Erf2p is directly involved in palmitate transfer. ERF2 and ERF4 are the first genes identified that encode a palmitoyltransferase for a Ras GTPase.     

Structure/function relationships responsible for the kinetic differences between human type 1 and type 2 3beta-hydroxysteroid dehydrogenase and for the catalysis of the type 1 activity

Two distinct genes encode the 93% homologous type 1 (placenta, peripheral tissues) and type 2 (adrenals, gonads) 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) in humans. Mutagenesis studies using the type 1 enzyme have produced the Y154F and K158Q mutant enzymes in the Y(154)-P-H(156)-S-K(158) motif as well as the Y269S and K273Q mutants from a second motif, Y(269)-T-L-S-K(273), both of which are present in the primary structure of the human type 1 3beta-HSD/isomerase. In addition, the H156Y mutant of the type 1 enzyme has created a chimera of the type 2 enzyme motif (Y(154)-P-Y(156)-S-K(158)) in the type 1 enzyme. The mutant and wild-type enzymes have been expressed and purified. The K(m) value of dehydroepiandrosterone is 13-fold greater, and the maximal turnover rate (K(cat)) is 2-fold greater for wild-type 2 3beta-HSD compared with the wild-type 1 3beta-HSD activity. The H156Y mutant of the type 1 enzyme has substrate kinetic constants for 3beta-HSD activity that are very similar to those of the wild-type 2 enzyme. Dixon analysis shows that epostane inhibits the 3beta-HSD activity of the wild-type 1 enzyme with 14-17-fold greater affinity compared with the wild-type 2 and H156Y enzymes. The Y154F and K158Q mutants exhibit no 3beta-HSD activity, have substantial isomerase activity, and utilize substrate with K(m) values similar to those of wild-type 1 isomerase. The Y269S and K273Q mutants have low, pH-dependent 3beta-HSD activity, exhibit only 5% of the maximal isomerase activity, and utilize the isomerase substrate very poorly. From these studies, a structural basis for the profound differences in the substrate and inhibition kinetics of the wild-type 1 and 2 3beta-HSD, plus a catalytic role for the Tyr(154) and Lys(158) residues in the 3beta-HSD reaction have been identified. These advances in our understanding of the structure/function of human type 1 and 2 3beta-HSD/isomerase may lead to the design of selective inhibitors of the type 1 enzyme not only in placenta to control the onset of labor but also in hormone-sensitive breast, prostate, and choriocarcinoma tumors to slow their growth.     

p27Kip1 is essential for the antiproliferative action of 1,25-dihydroxyvitamin D3 in primary,but not immortalized,mouse embryonic fibroblasts

1alpha,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits the growth of numerous cancer cell types. The intracellular proteins that mediate 1,25(OH)(2)D(3)-induced growth inhibition are poorly defined, although it is speculated that p21 and p27 are involved. We tested the requirement of p21 and p27 by treating primary wild-type, p21(-/-), and p27(-/-) mouse embryonic fibroblasts (MEFs) with 100 nm 1,25(OH)(2)D(3). In response to treatment, the wild-type and p21(-/-) MEFs exhibited 54 and 60% growth inhibition (p < 0.05), respectively, whereas the growth of p27(-/-) MEFs was unaffected. Western analyses indicated that p27 expression is induced by 1,25(OH)(2)D(3) treatment in wild-type and p21(-/-) MEFs. p21 expression is also induced by 1,25(OH)(2)D(3) treatment in wild-type and p27(-/-) MEFs, although the effect is less robust than for p27. Next, we spontaneously immortalized each MEF strain, which resulted in a gain of responsiveness to 1,25(OH)(2)D(3) by the p27(-/-) MEFs, as exhibited by 87% growth inhibition (p < 0.05). Both wild-type and p21(-/-) MEFs retained responsiveness (43 and 72% growth inhibition (p < 0.05), respectively). These data from primary and immortalized MEFs demonstrate that there are both p27-dependent and -independent pathways that mediate the antiproliferative action of 1,25(OH)(2)D(3).     

Characterization of mammalian eIF2A and identification of the yeast homolog

To begin the physical characterization of eukaryotic initiation factor (eIF) 2A, a translation initiation factor that binds Met-tRNA(i), tryptic peptides from rabbit reticulocyte eIF2A were analyzed to obtain amino acid sequence information. Sequences for 8 peptides were matched to three different expressed sequence tag clones. The sequence predicted for eIF2A is 585 amino acids. Matching of the cDNA sequence to the human genome revealed that the eIF2A mRNA is made up of 15 or 16 exons, and the gene is contained on chromosome 3. A homolog in Saccharomyces cerevisiae was identified, YGR054W, which is a non-essential gene. Hemagglutinin-tagged yeast eIF2A localizes on both 40 S and 80 S ribosomes. A knockout of both eIF2A and eIF5B yielded a "synthetically sick" yeast strain with a severe slow growth phenotype. The phenotype of this double mutant and the biochemical localization suggest that eIF2A participates in translation initiation. eIF2A does not appear to participate in re-initiation as the DeltaeIF2A strain shows the same level of GCN4 induction with amino acid starvation as seen in wild type yeast. The lack of any apparent phenotype in the DeltaeIF2A strain suggests that eIF2A functions in a minor pathway, perhaps internal initiation or in the translation of a small number of specific mRNAs.     

The hU3-55K protein requires 15.5K binding to the box B/C motif as well as flanking RNA elements for its association with the U3 small nucleolar RNA in Vitro

The 15.5K protein directly binds to the 5' stem-loop of the U4 small nuclear RNA, the small nucleolar (sno) RNA box C/D motif, and the U3 snoRNA-specific box B/C motif. The box B/C motif has also been shown to be essential for the association of the U3 small nucleolar ribonucleoprotein-specific protein hU3-55K. We therefore set out to determine how 15.5K and hU3-55K recognize the box B/C motif. By using an in vitro assembly assay, we show that hU3-55K effectively binds a sub-fragment of the U3 snoRNA surrounding the B/C motif that we have named the U3BC RNA. The association of hU3-55K with the U3BC RNA is dependent on the binding of 15.5K to the box B/C motif. The association of hU3-55K with the U3BC RNA was found to be also dependent on a conserved RNA structure that flanks the box B/C motif. Furthermore, we show that hU3-55K, a WD 40 repeat containing protein, directly cross-links to the U3BC RNA. Our data support a new structural model of the box B/C region of the U3 snoRNA in which the box B/C motif is base-paired to form a structure highly similar to that of both the U4 5' stem-loop and the box C/D motif.     

Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages

Surfactant protein-A (SP-A) plays multiple roles in pulmonary host defense, including stimulating bacterial phagocytosis by innate immune cells. Previously, SP-A was shown to interact with complement protein C1q. Our goal was to further characterize this interaction and elucidate its functional consequences. Radiolabeled SP-A bound solid-phase C1q but not other complement proteins tested. The lectin activity of SP-A was not required for binding to C1q. Because C1q is involved in bacterial clearance but alone does not efficiently enhance the phagocytosis of most bacteria, we hypothesize that SP-A enhances phagocytosis of C1q-coated antigens. SP-A enhanced by sixfold the percentage of rat alveolar macrophages in suspension that phagocytosed C1q-coated fluorescent beads. Furthermore, uptake of C1q-coated beads was enhanced when either beads or alveolar macrophages were preincubated with SP-A. In contrast, SP-A had no significant effect on the uptake of C1q-coated beads by alveolar macrophages adhered to plastic slides. We conclude that SP-A may serve a protective role in the lung by interacting with C1q to enhance the clearance of foreign particles.     

In vitro manipulations of vitamin C and vitamin E concentrations alter intracellular O2- production of hybrid striped bass (Morone chrysops x Morone saxatilis) head-kidney cells

To examine the mechanism by which vitamins C and E alter phagocyte function, a series of in vitro manipulations were conducted with cells isolated from the head-kidney of hybrid striped bass (average weight 680 g) fed a diet supplemented with minimum requirement levels of vitamins C and E for 2 weeks. Head-kidney phagocytes were cultured in media containing physiologically deficient (23 microM, adequate (45 microM) or excessive (182 microM) concentrations of vitamin C, and physiologically deficient (5 microM), adequate (9 microM) or excessive (32 microM) concentrations of vitamin E for 18 h. Following culture and stimulation, levels of reactive oxygen intermediates and hydrogen peroxide were determined. There were no effects of vitamin C or vitamin E concentrations on hydrogen peroxide or extracellular O2- generation. Intracellular O2- production, however, was significantly (P < or = 0.05) affected. When vitamin C was supplied at deficient levels to the medium, vitamin E elevated O2- production to levels not different from those of cells incubated with requirement levels of both vitamins. Similarly, when vitamin E was deficient in the media, vitamin C supplementation at requirement levels normalised intracellular O2- production. This data provides support for the presence of a vitamin C and vitamin E sparing mechanism in phagocytic head-kidney cells of hybrid striped bass and yield some insight into the mechanisms by which vitamin C and vitamin E function in immunomodulation.     

Unusual organic osmolytes in deep-sea animals: adaptations to hydrostatic pressure and other perturbants

Shallow-living marine invertebrates use free amino acids as cellular osmolytes, while most teleosts use almost no organic osmolytes. Recently we found unusual osmolyte compositions in deep-sea animals. Trimethylamine N-oxide (TMAO) increases with depth in muscles of some teleosts, skates, and crustaceans (up to 300 mmol/kg at 2900 m). Other deep-sea animals had high levels of (1). scyllo-inositol in echinoderms, gastropods, and polychaetes, (2). that polyol plus beta-alanine and betaine in octopods, (3). hypotaurine, N-methyltaurine, and unidentified methylamines in vestimentiferans from hydrothermal vents and cold seeps, and (4). a depth-correlated serine-phosphate osmolyte in vesicomyid clams from trench seeps. We hypothesize that some of these solutes counteract effects of hydrostatic pressure. With lactate dehydrogenase, actin, and pyruvate kinase, 250 mM TMAO (but not glycine) protected both ligand binding and protein stability against pressure. To test TMAO in living cells, we grew yeast under pressure. After 1 h at 71 MPa, 3.5 h at 71 MPa, and 17 h at 30 MPa, 150 mM TMAO generally doubled the number of cells that formed colonies. Sulfur-based osmolytes which are not correlated with depth, such as hypotaurine and thiotaurine, are probably involved in sulfide metabolism and detoxification. Thus deep-sea osmolytes may have at least two other roles beyond acting as simple compatible osmotica.     

Body zinc distribution profile during N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats at various levels of dietary zinc intake

Zinc distribution is apparently altered in breast cancer patients. It is unclear if this apparent zinc redistribution is a consequence of altered zinc nutrition or tissue-specific response to breast cancer. Our objectives were to assess effects of N-methyl-N-nitrosourea-treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis on body zinc-distribution profile in rats and to assess effects of dietary zinc intake on the body zinc-distribution profile during N-methyl-N-nitrosourea treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats. Female Sprague-Dawley rats were assigned to zinc-deficient (3 mg/kg diet) or zinc-adequate (31 mg/kg diet) ad libitum or pair-fed group. Rats were sham treated or N-methyl-N-nitrosourea treated (50 mg/kg body weight; Experiment 1 or 40 mg/kg body weight; Experiment 2) (n=6). In both experiments, the zinc concentration was significantly higher (6–19 times) in mammary tumor than in mammary gland. Tissue zinc concentration was essentially unaffected by N-methyl-N-nitrosourea treatment and tumor bearing, but was reduced by zinc deficiency in the bone, kidney, and liver. Overall, higher mammary tumor zinc concentration and absence of zinc redistribution during N-methyl-N-nitrosourea treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis, regardless of zinc intakes, indicates zinc accumulation in mammary tumors. Because zinc is essential for growth and cancer is characterized by uncontrolled growth, this zinc accumulation suggests an involvement of zinc in N-methyl-N-nitrosourea-induced rat mammary tumorigenesis.     

Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells

Interactions between microtubules (MTs) and filamentous actin (f-actin) are involved in directed cell locomotion, but are poorly understood. To test the hypothesis that MTs and f-actin associate with one another and affect each other's organization and dynamics, we performed time-lapse dual-wavelength spinning-disk confocal fluorescent speckle microscopy (FSM) of MTs and f-actin in migrating newt lung epithelial cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. Speckle analysis showed that MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. These results show that the movement and organization of f-actin has a profound effect on the dynamic organization of MTs in migrating cells, and suggest that MTs and f-actin bind to one another in vivo.