Import and export of nuclear proteins: focus on the nucleocytoplasmic movements of two different species of mammalian estrogen receptor

There is a wealth of information regarding the import and export of nuclear proteins in general. Nevertheless, the available data that deals with the nucleocytoplasmic movement of steroid hormone receptors remains highly limited. Some research findings reported during the past five years have succeeded in identifying proteins related to the movement of estrogen receptor alpha from the cytoplasm to the nucleus. What is striking in these findings is the facilitatory role of estradiol in the transport process. A similar conclusion has been drawn from the studies on the plasma membrane-to nucleus movement of the alternative form of estrogen receptor, the non-activated estrogen receptor (naER). The internalization of naER from the plasma membrane takes place only in the presence of estradiol. While the gene regulatory functions of ER alpha appear to get terminated following its ubiquitinization within the nucleus, the naER, through its deglycosylated form, the nuclear estrogen receptor II (nER II) continues to remain functional even beyond its existence within the nucleus. Recent studies have indicated the possibility that the estrogen receptor that regulates the nucleo cytoplasmic transport of m RNP is the nERII. This appears to be the result of the interaction between nERII and three proteins belonging to a group of small nuclear ribonucleo proteins (snRNP). The interaction of nERII with two of this protein appears to activate the inherent Mg2+ ATPase activity of the complex, which leads to the exit of the RNP through the nuclear pore complex.     

Estrogen directly down-regulates the bone-resorbing activity of mature osteoclasts through nuclear estrogen receptor α

The decrease in estrogen level that follows the onset ofmenopause causes rapid bone loss, resulting in osteoporosis.However, the mechanism remains unclear, especially concerningthe regulation of bone-resorbing osteoclasts. Here we analyzedthe function of estrogen and its receptor in matureosteoclasts. We found that estrogen directly inhibitedbone-resorption by purified rabbit mature-osteoclasts.Moreover, using a RT-PCR technique, we report that nuclearestrogen receptor (ER) but not ER is expressed in mature osteoclasts. The antisense oligodeoxynucleotide for ER inhibited the reductionin osteoclastic bone-resorbing activity caused by estrogen. We conclude that in part estrogen directly inhibits the bone-resorbing activity of mature osteoclasts through the ER.     

Basal Forebrain Cholinergic Immunolesion by 192IgG-Saporin: Evidence for a Presynaptic Location of Subpopulations of α2-and β-Adrenergic as Well as 5-HT2A Receptors on Cortical Cholinergic Terminals

To study whether the changes in cortical noradrenergic and serotonergic mechanisms observed in patients with Alzheimer's disease are the consequence of reduced cortical cholinergic activity, a novel cholinergic immunotoxin (conjugate of the monoclonal antibody 192IgG against the low-affinity nerve growth factor receptor with the cytotoxic protein saporin, 192IgG-saporin) was used to produce a specific and selective loss of cholinergic cells in rat basal forebrain nuclei. To correlate the responses to cholinergic immunolesion in cholinoceptive cortical target regions with cholinergic hypoactivity, quantitative receptor autoradiography to measure adrenoceptors and 5-hydroxytryptamine (5-HT) receptor subtypes, and histochemistry to estimate acetylcholinesterase activity, were performed in adjacent brain sections. ₁-adrenoceptor and 5-HT_1A receptor binding were not affected by cholinergic immunolesion in any of the cortical and hippocampal regions studied. However, cholinergic immunolesion resulted in significantly reduced ₂-and -adrenoceptor as well as 5-HT_2A receptor binding in a number cortical and hippocampal regions displaying a reduced activity of acetylcholinesterase, already detectable seven days after a single injection of 192IgG-saporin and persisting up to three months post lesion without any significant recovery. The data suggest that at least a subpopulation of ₂-and -adrenoceptor as well 5-HT_2A receptor subtype is present on cortical and hippocampal cholinergic terminals originating in the basal forebrain. The lesion-induced receptor changes suggest that the alterations in cortical 5-HT₂ receptor binding observed in patients with Alzheimer's disease might be secondary to the cholinergic deficits.     

SpαI/65 hereditary elliptocytosis in Calabria (southern Italy)

The ^I/65 variant of spectrin has been described in black people, in North Africans and recently in two southern Italian families. This variant is associated in the heterozygous state with mild Hereditary Elliptocytosis (HE) and the molecular basis of the defect is invariably the duplication of TTG at codon 154 of the spectrin gene. The present study reports the identification of five Calabrian families with SP^I/65 HE and their distribution in the population.     

A comparative study of enzyme activity variation between α-glycerophosphate and alcoholdehydrogenases in Drosophila melanogaster

An intraspecific comparison of -glycerophosphate (-GPDH: E.C.1.1.1.8) and alcohol dehydrogenase (ADH: E.C.1.1.1.1) enzyme activity levels was carried out in Drosophila melanogaster. The results indicate that (1) -GPDH is a relatively conservative and ADH a relatively variable enzyme system with regard to structurally determined activity variation but that (2) the conservative nature of -GPDH activity variation does not extend to the intra-genotypic level. The results are consistent with the view that different kinds of selective pressures are being exerted on the enzyme's structural and modifier gene loci.     

Reversed micelles recognize an active protein

Summary The extraction behavior of native and heated-denatured -chymotrypsin has been investigated with two different reversed micellar systems. A large difference in the degree of extraction was observed for the native relative to the denatured -chymotrypsin. In particular, mixed reversed micelles formulated with DOLPA (dioleyl phosphoric acid) and AOT show a high selectivity for the active -chymotrypsin.     

Nuclear estrogen receptor II (nER-II) is involved in the estrogen-dependent ribonucleoprotein transport in the goat uterus I. Localization of nER-II in snRNP.

Exposure of goat uterine nuclei to estradiol in vitro results in an immediate exit of ribonucleoproteins (RNP) from the nuclei to the medium. This RNP exit appears to be mediated by an estrogen receptor localized in small nuclear ribonucleoproteins containing U1 and U2 snRNA. Available evidence indicates that the estrogen receptor involved is not the ERalpha, but an alternative form, which is also a 66 kDa protein. This is the nuclear estrogen receptor II (nER-II) that has no DNA-binding capacity. The transport is estrogen-specific since non-estrogenic steroids do not stimulate the transport of the RNP where the receptor is localized.     

Efficient determination of angles subtended by Cα-Hα and N-HN vectors in proteins via dipole-dipole cross-correlation§

The angle CH,NHN subtended by the internuclear vectors 13C-H and 15N-HN in doubly-labeled proteins can be determined by observing the effect of cross-correlation between the dipolar interactions on zero- and double-quantum coherences involving 13C and 15N. Two complementary 2D experiments with the appearance of 15N-HN correlation spectra yield signal intensities that depend on the rate of interconversion through cross-correlated relaxation of in-phase and doubly antiphase zero- and double-quantum coherences. The ratio of the signal intensities in the two experiments bears a simple relationship to the cross-correlation rate, and hence to the angle CH,NHN. Assuming planarity of the peptide bond, the dihedral angle (between C and C) can be determined from the knowledge of CH,NHN. The experiments are very time-effective and provide good sensitivity and excellent spectral resolution.     

Primary structure of an agonist binding subunit of the nicotinic acetylcholine receptor from bovine adrenal chromaffin cells

Activation by acetylcholine of a nicotinic acetylcholine receptor on the membrane of bovine chromaffin cells leads to membrane depolarization and to the subsequent triggering of catecholamine secretion. It is evident that acetylcholine receptors play a central role in the initial phase of the secretion process and, therefore, an extensive characterization of their molecular components and properties is of fundamental interest. With this intention, we have screened bovine adrenal medullary cDNA libraries with a probe coding for a fragment of the rat muscle acetylcholine receptor subunit. Several cDNA clones were isolated. The longest cDNA had an open reading frame encoding a 495-amino acid protein with a molecular weight of 56,911. The deduced primary structure contains features that indicate that the encoded protein is an or acetylcholine binding subunit, and, in fact, it manifests significant sequence similarity to previously cloned subunits. Sequence identity is particularly high with the 3 subunit, which is expressed in the rat pheochromocytoma PC12 cell line and in several brain areas, and consequently, it is considered a component of a neuronal acetylcholine receptor. Accordingly, the present results suggest that the agonist binding subunit of the nicotinic acetylcholine receptor from bovine chromaffin cells is an 3-type subunit, corroborating previous immunological and pharmacological evidence for the presence of a neuronal nicotinic receptor in chromaffin cells.Abbreviations used nAChR nicotinic acetylcholine receptor - SDS sodium dodecyl sulfate - SSC 0.15 M NaCl and 0.015 M sodium citrate - kb kilobases - bp base pairs     

Purification of secreted α-amylases by immunoaffinity chromatography with cross-reactive antibody

Two isozymes of rice -amylases expressed and secreted by recombinant yeast were purified by immunoaffinity chromatography by using cross-reactive antibody. Antibodies raised against partially purified barley -amylase adsorbed rice -amylases in fermentation broth by a cross-reaction. By use of these antibodies as ligands, rice -amylases were concentrated and purified to a high degree in one-step immunoaffinity chromatography. Because of the differences in the contaminating impurities between the barley -amylase (antigen) from barley malt and rice -amylases (target protein) secreted from yeast, the high purity of eluted -amylases was attained without the use of highly purified antigen for immunization. Utilization of cross-reactive antibodies in immunoaffinity chromatography is useful for the purification of recombinant proteins in the absence of a sufficient amount and high enough purity of the target proteins to be purified.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: a consequence of the formation of "native-like conformation".

The influence of n-propanol on the overall -helical conformation of -globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of -helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of -helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of -globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The -helical conformation induced into - and -globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the -helical conformation of both - and -chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced -helical conformation of globins and the -helical conformation of - and -chains. A cross-correlation of the n-propanol induced increase in the fluorescence of -globin with the corresponding increase in the -helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the -helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a native-like structure. The induction of -helical conformation into these proteins in the presence of n-propanol and the consequent generation of native-like conformation is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an -helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume -helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high -helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of -helical proteins. Consistent with this concept, the induction of -helical conformation into shorter polypeptide fragments of 30 residues, (e.g., _1-30, which exists in an -helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Effects of mutations in the N terminal region of the yeast G protein α-subunit Gpa1p on signaling by pheromone receptors

The sites and modes of interaction between G protein-coupled receptors and their cognate heterotrimeric G proteins remain poorly defined. The C-terminus of the G subunit is the best established site of contact of G proteins with receptors, but structural analyses and crosslinking studies suggest the possibility of interactions at the N-terminus of G as well. We screened for mutations in the N-terminal region of the G subunit encoded by the yeast GPA1 gene that specifically affect the ability of the G protein to be activated by the yeast -mating factor receptor. The screen led to identification of substitutions of glutamine or proline for Leu18 of Gpa1p that reduce the response to the pheromones -factor and a-factor without affecting cellular levels of the subunit or its ability to interact with and subunits. The mutations do not appear to affect the intrinsic ability of the G protein to be converted to the activated state. The low yield of different mutations with this phenotype indicates either that the N-terminal segment of the yeast G subunit does not undergo extensive interactions with the -factor receptor, or that this region can not be altered without detrimental effects upon the formation of G protein trimers.Communicated by D. Y. Thomas     

Characterization,subcellular localization and nuclear targeting of casein kinase 2 from Zea mays

We have isolated and characterized the genomic clone of maize casein kinase 2 (CK2) subunit using the previously described CK2-1 cDNA clone as a probe. The genomic clone is 7.5 kb long and contains 10 exons, separated by 9 introns of different size, two larger than 1.5 kb and the others around 100–150 bp. The sequence of the exons is 100% homologous to the sequence of the CK2-1 cDNA. Southern hybridization of total genomic DNA from maize embryos with CK2 cDNA indicated that the CK2-1 gene is part of a multigenic family. We also isolated a new embryo cDNA clone coding for an CK2-2 subunit. We studied the regulation of the enzyme in embryos at the mRNA level, at the protein level and by activity testing. By using immunocytochemistry the CK2 protein was localized in several types of cells of mature embryos. Particularly strong signals were visible in the cytoplasm of epidermis and meristematic cells. Decoration of nuclei of root cortex and scutellum cells was also observed suggesting that CK2 can shift from the cytoplasm into nuclei in specific cell types. We examined whether CK2 contained specific protein domains which actively target the protein to the nucleus by using in-frame fusions of the maize CK2 subunit to the reporter gene encoding -glucuronidase (GUS) which were assayed in transiently transformed onion epidermal cells. Analysis of chimeric constructs identified one region containing a nuclear localization signal (NLS) that is highly conserved in other CK2 proteins.     

Sequence analysis of 22 kDa-like α-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements

Several genomic and cDNA clones encoding the 22 kDa-like -coixin, the -prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like -coixin genes designated -3A, -3B and -3C were found in the 15 kb -3 genomic clone. The -3A and -3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the -3B gene, suggesting that the three -coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication.Comparison of the deduced amino acid sequences of -coixin clones with the published sequences of 22 kDa -zein and 22 kDa-like -kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15–20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like -prolamins and the 19 kDa -zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins.Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5 and 3 flanking regions of -3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. –300 prolamin box are present at conserved positions in -3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in -3B that occupies approximately the same positions as those identified for the 22 kDa -zein and -kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes.The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like -prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.     

Membrane associated estrogen receptors and related proteins: Localization at the plasma membrane and the endoplasmic reticulum

The female sex steroid, estradiol 17, mediates its effect through its association with estrogen receptor present in the target cell. So far the major emphasis has been given to the genomic actions of the hormone mediated by the nuclear estrogen receptors. Recent years have seen a shift in the ideas revealing the existence of estradiol binding entities both in the plasma membrane and the endoplasmic reticulum. Though the true identity of this membrane associated receptors is far from being known, a functional role for the same have been implicated both at the genomic as well as the non-genomic level. The major focus of the review is to highlight the existence of membrane associated estrogen receptors and receptor-related proteins and the functional roles played by some of them. The signalling events exerted by this class of membrane associated estrogen receptor could partly explain the physiological significance of estrogen in cardiovascular disease, osteoporosis and breast cancer as well as the molecular mechanism associated with xenoestrogen action.     

Conditional Over-Expression of Estrogen Receptor Alpha in a Transgenic Mouse Model

Attempts to delineate the mechanisms of estrogen action have promoted the creation of several estrogen receptor alpha (ER) mouse models in the past decade. These traditional models are limited by the fact that the receptors are either absent or present throughout all stages of development. The purpose of this work was to develop a conditional transgenic model that would provide an in vivo method of controlling the spatial and temporal regulation of ER expression. The tetracycline responsive system was utilized. Three lines of transgenic mice carrying a transgene composed of the coding sequence for murine ER placed under the regulatory control of a tet operator promoter (tet-op) were generated. These three lines of tet-op-mER mice were each mated to an established line of transgenic mice expressing a tetracycline-dependent transactivator protein (tTA) from the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Double transgenic MMTV-tTA/tet-op-mER mice were produced. All three lines demonstrated dominant gain of ER shown by RT-PCR, immunoprecipitation, and immunohistochemistry. Transgene-specific ER was expressed in numerous tissues including the mammary gland, salivary gland, testis, seminal vesicle, and epididymis. Expression was silenced by administration of doxycycline in the drinking water. This model can be utilized to evaluate the consequences of ER dominant gain in targeted tissues at specific times during development. In this study dominant gain of ER was associated with a reduction in epididymal/vas deferens and seminal vesicle weights consistent with the proposed action of ER on fluid transport in the male reproductive tract. Combining this model with other dominant gain and gene knockout mouse models will be useful for testing effects of ER action in combination with specific gene products and to evaluate if developmental and stage-specific expression of ER can rescue identified phenotypes in gene knockout mice.     

Expression of enzymatically active,recombinant barley α-glucosidase in yeast and immunological detection of α-glucosidase from seed tissue

An -glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae -factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant -glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5–6.3 with an optimum at pH 4.3, classifying the enzyme as an acid -glucosidase. The enzyme had a K_m of 1.88 mM and V_max of 0.054 µmol/min on maltose. The recombinant -glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley -glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa -glucosidase became prominent. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in -glucosidase enzyme activity.Abbreviations: AGL, barley seed -glucosidase; rAGL, recombinant barley seed -glucosidase; BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; GA, gibberellic acid; UTR, untranslated region.     

Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF2) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 kinases. Further, only wheat wild-type eIF2 is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 kinases. A truncated version of wild-type wheat eIF2 containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 species and eIF2 kinases and support the presence of a plant eIF2 phosphorylation pathway.