Comparative Mg(2+)-dependent sequential covalent binding stoichiometries of 3'-O-(4-benzoyl)benzoyl adenosine 5'-diphosphate of MF1, TF1, and the alpha 3 beta 3 core complex of TF1. The binding change motif is independent of the F1 gamma delta epsilon subunits.

PubMed

Aloise, P; Kagawa, Y; Coleman, P S

1991-06-05

Three F1 preparations, the beef heart (MF1) and thermophilic bacterium (TF1) holoenzymes, and the alpha 3 beta 3 "core" complex of TF1 reconstituted from individually expressed alpha and beta subunits, were compared as to their kinetic and binding stoichiometric responses to covalent photoaffinity labeling with BzATP and BzADP (+/- Mg2+). Each enzyme displayed an enhanced pseudo-first order rate of photoinhibition and one-third of the sites covalent binding to a catalytic site for full inhibition, plus, but not minus Mg2+. Titration of near stoichiometric [MgBzADP]/[F1] ratios during photolysis disclosed two sequential covalent binding patterns for each enzyme; a high affinity binding corresponding to unistoichiometric covalent association concomitant with enzyme inhibition, followed by a low affinity multisite-saturating covalent association. Thus, in the absence of the structural asymmetry inducing gamma delta epsilon subunits of the holoenzyme, the sequential binding of nucleotide at putative catalytic sites on the alpha 3 beta 3 complex of any F1 appears sufficient to effect binding affinity changes. With MF1, final covalent saturation of BzADP-accessible sites was achieved with 2 mol of BzADP/mol of enzyme, but with TF1 or its alpha 3 beta 3 complex, saturation required 3 mol of BzADP/mol of enzyme. Such differential final labeling stoichiometries could arise because of the endogenous presence of 1 nucleotide already bound to one of the 3 potential catalytic sites on normally prepared MF1, whereas TF1, possessing no endogenous nucleotide, has 3 vacant BzADP-accessible sites. Kinetics measurements revealed that regardless of the incremental extent of inhibition of the TF1 holoenzyme by BzADP during photolysis, the two higher apparent Km values (approximately 1.5 x 10(-4) and approximately 10(-3) M, respectively) of the progressively inactivated incubation are unchanged relative to fully unmodified enzyme. As reported for BzATP (or BzADP) and MF1 (Ackerman, S.H., Grubmeyer, C., and Coleman, P.S. (1987) J. Biol. Chem. 262, 13765-13772), this supports the fact that the photocovalent inhibition of F1 is a one-hit one-kill phenomenon. Isoelectric focusing gels revealed that [3H]BzADP covalently modifies both TF1 and MF1 exclusively on the beta subunit, whether or not Mg2+ is present. A single 19-residue [3H]BzADP-labeled peptide was resolved from a tryptic digest of MF1, and this peptide corresponded with the one believed to contain at least a portion of the beta subunit catalytic site domain (i.e. beta Ala-338----beta Arg-356).

Direct interaction of the inhibitory gamma-subunit of Rod cGMP phosphodiesterase (PDE6) with the PDE6 GAFa domains.

PubMed

Muradov, Khakim G; Granovsky, Alexey E; Schey, Kevin L; Artemyev, Nikolai O

2002-03-26

Retinal rod and cone cGMP phosphodiesterases (PDE6 family) function as the effector enzyme in the vertebrate visual transduction cascade. The activity of PDE6 catalytic subunits is controlled by the Pgamma-subunits. In addition to the inhibition of cGMP hydrolysis at the catalytic sites, Pgamma is known to stimulate a noncatalytic binding of cGMP to the regulatory GAFa-GAFb domains of PDE6. The latter role of Pgamma has been attributed to its polycationic region. To elucidate the structural basis for the regulation of cGMP binding to the GAF domains of PDE6, a photoexcitable peptide probe corresponding to the polycationic region of Pgamma, Pgamma-21-45, was specifically cross-linked to rod PDE6alphabeta. The site of Pgamma-21-45 cross-linking was localized to Met138Gly139 within the PDE6alpha GAFa domain using mass spectrometric analysis. Chimeras between PDE5 and cone PDE6alpha', containing GAFa and/or GAFb domains of PDE6alpha' have been generated to probe a potential role of the GAFb domains in binding to Pgamma. Analysis of the inhibition of the PDE5/PDE6alpha' chimeras by Pgamma supported the role of PDE6 GAFa but not GAFb domains in the interaction with Pgamma. Our results suggest that a direct binding of the polycationic region of Pgamma to the GAFa domains of PDE6 may lead to a stabilization of the noncatalytic cGMP-binding sites.

Mutation in rod PDE6 linked to congenital stationary night blindness impairs the enzyme inhibition by its gamma-subunit.

PubMed

Muradov, Khakim G; Granovsky, Alexey E; Artemyev, Nikolai O

2003-03-25

Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the vertebrate visual transduction cascade. The activity of rod PDE6 catalytic alpha- and beta-subunits is blocked in the dark by two inhibitory Pgamma-subunits. The inhibition is released upon light-stimulation of photoreceptor cells. Mutation H258N in PDE6beta has been linked to congenital stationary night blindness (CSNB) in a large Danish family (Rambusch pedigree) (Gal, A., Orth, U., Baehr, W., Schwinger, E., and Rosenberg, T. (1994) Nat. Genet. 7, 64-67.) We have analyzed the consequences of this mutation for PDE6 function using a Pgamma-sensitive PDE6alpha'/PDE5 chimera, Chi16. Biochemical analysis of the H257N mutant, an equivalent of PDE6betaH258N, demonstrates that this substitution does not alter the ability of chimeric PDE to dimerize or the enzyme's catalytic properties. The sensitivity of H257N to a competitive inhibitor zaprinast was also unaffected. However, the mutant displayed a significant impairment in the inhibitory interaction with Pgamma, which was apparent from a approximately 20-fold increase in the K(i) value (46 nM) and incomplete maximal inhibition. The inhibitory defect of H257N is not due to perturbation of noncatalytic cGMP binding to the PDE6alpha' GAF domains. The noncatalytic cGMP-binding characteristics of the H257N mutant were similar to those of the parent PDE6alpha'/PDE5 chimera. Since rod PDE6 in the Rambusch CSNB is a catalytic heterodimer of the wild-type PDE6alpha and mutant PDE6beta, Chi16 and H257N were coexpressed, and a heterodimeric PDE, Chi16/H257N, was isolated. It displayed two Pgamma inhibitory sites with the K(i) values of 5 and 57 nM. Our results support the hypothesis that mutation H258N in PDE6beta causes CSNB through incomplete inhibition of PDE6 activity by Pgamma, which leads to desensitization of rod photoreceptors.

Partial reconstitution of photoreceptor cGMP phosphodiesterase characteristics in cGMP phosphodiesterase-5.

PubMed

Granovsky, A E; Artemyev, N O

2001-06-15

Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serve as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight association with the inhibitory gamma-subunits (Pgamma). The Pgamma block is removed in the light-activated PDE6 by the visual G protein, transducin. Transducin-activated PDE6 exhibits an exceptionally high catalytic rate of cGMP hydrolysis ensuring high signal amplification. To identify the structural determinants for the inhibitory interaction with Pgamma and the remarkable cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristics by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE. PDE5 is insensitive to Pgamma and has a more than 100-fold lower k(cat) for cGMP hydrolysis. Our mutational analysis of chimeric PDE5/PDE6alpha' enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6alpha') with Pgamma is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met(758), Phe(777), and Phe(781). The maximal catalytic rate of PDE5 was enhanced by at least 10-fold with substitutions of PDE6alpha'-specific glycine residues for the corresponding PDE5 alanine residues, Ala(608) and Ala(612). The Gly residues are adjacent to the highly conserved metal binding motif His-Asn-X-X-His, which is essential for cGMP hydrolysis. Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis.

Forskolin-mediated BeWo cell fusion involves down-regulation of miR-92a-1-5p that targets dysferlin and protein kinase cAMP-activated catalytic subunit alpha.

PubMed

Dubey, Richa; Malhotra, Sudha S; Gupta, Satish K

2018-06-01

To study the role of miRNA(s) during trophoblastic BeWo cell fusion. Changes in miRNA(s) profile of BeWo cells treated with forskolin were analyzed using Affymetrix miRNA microarray platform. Down-regulated miRNA, miR-92a-1-5p, was overexpressed in BeWo cells followed by forskolin treatment to understand its relevance in the process of BeWo cell fusion by desmoplakin I+II staining and hCG secretion by ELISA. Predicted targets of miR-92a-1-5p were also confirmed by qRT-PCR/Western blotting. The miRNA profiling of BeWo cells after forskolin (25 μmol/L) treatment identified miR-92a-1-5p as the most significantly down-regulated miRNA both at 24 and 48 hours time points. Overexpression of miR-92a-1-5p in these cells led to a significant decrease in forskolin-mediated cell fusion and hCG secretion. miRNA target prediction software, TargetScan, revealed dysferlin (DYSF) and protein kinase cAMP-activated catalytic subunit alpha (PRKACA), as target genes of miR-92a-1-5p. Overexpression of miR-92a-1-5p in BeWo cells showed reduction in forskolin-induced transcripts for DYSF and PRKACA. Further, reduction in DYSF (~2.6-fold) at protein level and PRKACA-encoded protein kinase A catalytic subunit alpha (PKAC-α; ~1.6-fold) were also observed. These observations suggest that miR-92a-1-5p regulates forskolin-mediated BeWo cell fusion and hCG secretion by regulating PKA signaling pathway and dysferlin expression. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

LKB1, an upstream AMPK kinase, regulates glucose and lipid metabolism in cultured liver and muscle cells.

PubMed

Imai, Kenta; Inukai, Kouichi; Ikegami, Yuichi; Awata, Takuya; Katayama, Shigehiro

2006-12-22

LKB1 is a 50 kDa serine/threonine kinase that phosphorylates and activates the catalytic subunit of AMPK at its T-loop residue Thr 172. We prepared adenoviruses expressing the constitutive active (wild-type) form (CA) or dominant negative (kinase inactive, D194A mutant) form (DN) of LKB1 and overexpressed these proteins in cultured myotubes (C2C12 cells) and rat hepatoma cells (FAO cells). When analyzed by immunoblotting with the antibody against Thr172-phosphorylated AMPK, the phosphorylation of AMPK was increased (2.5-fold) and decreased (0.4-fold) in cells expressing CA and DN LKB1, respectively, as compared with Lac-Z expressing control cells. Immunoprecipitation experiments, using isoform-specific antibody, revealed these alterations of AMPK phosphorylation to be attributable to altered phosphorylation of AMPK alpha2, but not alpha1 catalytic subunits, strongly suggesting the alpha2 catalytic subunit to be the major substrate for LKB1 in mammalian cells. In addition, adiponectin or AICAR-stimulated AMPK phosphorylation was inhibited by overexpression of DN LKB1, while phenformin-stimulated phosphorylation was unaffected. These results may explain the difference in AMPK activation mechanisms between AMP and phenformin, and also indicate that AMPK phosphorylation by LKB1 is involved in AMP-stimulated AMPK activation. As a downstream target for AMPK, AICAR-induced glucose uptake and ACCbeta phosphorylation were found to be significantly reduced in DN LKB1 expressing C2C12 cells. The expression of key enzymes for gluconeogenesis, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was also dependent on LKB1 activities in FAO cells. These results demonstrate that LKB1 is a crucial regulator of AMPK activation in muscle and liver cells and, therefore, that LKB1 activity is potentially of importance to our understanding of glucose and lipid metabolism.

Mechanisms of the HRSL3 tumor suppressor function in ovarian carcinoma cells.

PubMed

Nazarenko, Irina; Schäfer, Reinhold; Sers, Christine

2007-04-15

HRSL3 (also known as H-REV107-1) belongs to a class II tumor suppressor gene family and is downregulated in several human tumors including ovarian carcinomas. To unravel the mechanism of HRSL3 tumor suppressor action, we performed a yeast two-hybrid screen and identified the alpha-isoform of the regulatory subunit A of protein phosphatase 2A (PR65alpha) as a new interaction partner of HRSL3. Interaction between HRSL3 and PR65alpha was confirmed in vitro and by co-immunoprecipitation in mammalian cells. We demonstrate that HRSL3 binds to the endogenous PR65alpha, thereby partially sequestering the catalytic subunit PR36 from the PR65 protein complex, and inhibiting PP2A catalytic activity. Furthermore, binding of HRSL3 to PR65 induces apoptosis in ovarian carcinoma cells in a caspase-dependent manner. Using several mutant HRSL3 constructs, we identified the N-terminal proline-rich region within the HRSL3 protein as the domain that is relevant for both binding of PR65alpha and induction of programmed cell death. This suggests that the negative impact of HRSL3 onto PP2A activity is important for the HRSL3 pro-apoptotic function and indicates a role of PP2A in survival of human ovarian carcinomas. The analysis of distinct PP2A target molecules revealed PKCzeta as being involved in HRSL3 action. These data implicate HRSL3 as a signaling regulatory molecule, which is functionally involved in the oncogenic network mediating growth and survival of ovarian cancer cells.

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

PubMed

Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

1999-03-05

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

PubMed Central

Mayer-Jaekel, R E; Baumgartner, S; Bilbe, G; Ohkura, H; Glover, D M; Hemmings, B A

1992-01-01

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner. Images PMID:1320961

Highly conserved small subunit residues influence rubisco large subunit catalysis.

PubMed

Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster.

PubMed

Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O; Dobritsa, Anna A; Evstafieva, Alexandra G; Boldyreff, Brigitte; Issinger, Olaf-Georg; Gvozdev, Vladimir A

2002-03-01

An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta)tes-beta-galactosidase fusion protein driven by the CK2(beta)tes promoter was found in transgenic flies at postmitotic stages of spermatogenesis. Examination of biochemical characteristics of a recombinant CK2(beta)tes protein expressed in Escherichia coli revealed properties similar to those of CK2beta: (a) CK2(beta)tes protein stimulates CK2alpha catalytic activity toward synthetic peptide; (b) it inhibits phosphorylation of calmodulin and mediates stimulation of CK2alpha by polylysine; (c) it is able to form (CK2(beta)tes)2 dimers, as well as (CK2alpha)2(CK2(beta)tes)2 tetramers. Using the yeast two-hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two tissue-specific regulatory subunits of CK2 which might serve to provide CK2 substrate recognition during spermatogenesis.

Binding-dependent disorder-order transition in PKI alpha: a fluorescence anisotropy study.

PubMed

Hauer, J A; Taylor, S S; Johnson, D A

1999-05-25

The conformational flexibility of peptidyl ligands may be an essential element of many peptide-macromolecular interactions. Consequently, the alpha-carbonyl backbone flexibility of the 8 kDa protein kinase inhibitor (PKI alpha) peptide of cAMP-dependent protein kinase (cAPK) free in solution and bound to cAPK was assessed by time-resolved fluorescence anisotropy. Specifically, three full-length, single-site PKI alpha mutants (V3C, S28C, and S59C) were prepared, and fluorescein iodoacetamide (FI) was selectively conjugated to the side chains of each substituted cysteine. The time-resolved anisotropy decay profiles of the labeled mutants were well fit to a model-free nonassociative biexponential equation. Free in solution, the three labeled proteins had very similar anisotropy decays arising primarily from local alpha-carbonyl backbone movements. Only a small fraction of the anisotropy decay was associated with slower, whole-body tumbling, confirming that PKI alpha is highly disordered at all three locations. Complexation of the mutants with the catalytic (C) subunit of cAPK decreased the rate of whole-body tumbling for all three mutants. The effects on the rapid decay processes, however, were dependent upon the site of conjugation. The anisotropy decay profiles of both FI-V3C- and FI-S28C-PKI alpha were associated with significantly reduced contributions from the fast decay processes, while that of FI-S59C-PKI alpha was largely unaffected by binding to the C-subunit. The results suggest that the cAPK-binding domain of PKI alpha extends from the its N-terminus to residues beyond Ser28 but does not include the segment around Ser59, which is still part of a highly flexible domain when bound to the C-subunit.

Two hydrophobic subunits are essential for the heme b ligation and functional assembly of complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli.

PubMed

Nakamura, K; Yamaki, M; Sarada, M; Nakayama, S; Vibat, C R; Gennis, R B; Nakayashiki, T; Inokuchi, H; Kojima, S; Kita, K

1996-01-05

Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits encoded by the sdhCDAB operon. Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein) to the cytoplasmic membrane and are believed to be the components of cytochrome b556 in E. coli complex II. In the present study, to elucidate the role of two hydrophobic subunits in the heme b ligation and functional assembly of complex II, plasmids carrying portions of the sdh gene were constructed and introduced into E. coli MK3, which lacks succinate dehydrogenase and fumarate reductase activities. The expression of polypeptides with molecular masses of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, indicating that sdhC encodes the large subunit (cybL) and sdhD the small subunit (cybS) of cytochrome b556. An increase in cytochrome b content was found in the membrane when sdhD was introduced, while the cytochrome b content did not change when sdhC was introduced. However, the cytochrome b expressed by the plasmid carrying sdhD differed from cytochrome b556 in its CO reactivity and red shift of the alpha absorption peak to 557.5 nm at 77 K. Neither hydrophobic subunit was able to bind the catalytic portion to the membrane, and only succinate dehydrogenase activity, not succinate-ubiquinone oxidoreductase activity, was found in the cytoplasmic fractions of the cells. In contrast, significantly higher amounts of cytochrome b556 were expressed in the membrane when sdhC and sdhD genes were both present, and the catalytic portion was found to be localized in the membrane with succinate-ubiquitnone oxidoreductase and succinate oxidase activities. These results strongly suggest that both hydrophobic subunits are required for heme insertion into cytochrome b556 and are essential for the functional assembly of E. coli complex II in the membrane. Accumulation of the catalytic portion in the cytoplasm was found when sdhCDAB was introduced into a heme synthesis mutant, suggesting the importance of heme in the assembly of E. coli complex II.

Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cocozaki, Alexis I.; Ramia, Nancy F.; Shao, Yaming

Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3 {angstrom} crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two {alpha}-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases andmore » bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.« less

Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase [alpha

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Jia; Yang, Yuting; Wan, Ke

Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, ourmore » structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase {alpha} (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.« less

Site directed mutagenesis of the heme axial ligands of cytochrome b559 affects the stability of the photosystem II complex.

PubMed Central

Pakrasi, H B; De Ciechi, P; Whitmarsh, J

1991-01-01

Cytochrome (cyt) b559, an integral membrane protein, is an essential component of the photosystem II (PSII) complex in the thylakoid membranes of oxygenic photosynthetic organisms. Cyt b559 has two subunits, alpha and beta, each with one predicted membrane spanning alpha-helical domain. The heme cofactor of this cytochrome is coordinated between two histidine residues. Each of the two subunit polypeptides of cyt b559 has one His residue. To investigate the influence of these His residues on the structure of cyt b559 and the PSII complex, we used a site directed mutagenesis approach to replace each His residue with a Leu residue. Introduction of these missense mutations in the transformable unicellular cyanobacterium, Synechocystis 6803, resulted in complete loss of PSII activity. Northern blot analysis showed that these mutations did not affect the stability of the polycistronic mRNA that encompasses both the psbE and the psbF genes, encoding the alpha and the beta subunits, respectively. Moreover, both of the single His mutants showed the presence of the alpha subunit which was 1.5 kd smaller than the same polypeptide in wild type cells. A secondary effect of such a structural change was that D1 and D2, two proteins that form the catalytic core (reaction center) of PSII, were also destabilized. Our results demonstrate that proper axial coordination of the heme cofactor in cyt b559 is important for the structural integrity of the reaction center of PSII. Images PMID:1904816

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jack Preiss

Conversion of the Potato tuber ADP-glucose Pyrophopshorylase Regulatory Subunit into a Catalytic Subunit. ADP-glucose synthesis, a rate-limiting reaction in starch synthesis, is catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). The enzyme in plants is allosterically activated by 3-phosphoglycerate (3PGA) and inhibited by inorganic phosphate (Pi) and is composed of two subunits as a heterotetramer, a2b2. Subunit a is the catalytic subunit and subunit b is designated as the regulatory subunit.The b subunit increases the affinty of the activator for the catalytic subunit. Recent results have shown that the subunits are derived from the same ancestor subunit as the regulatory subunit canmore » be converted to a catalytically subunit via mutation of just two amino acids. Lys44 and Thr54 in the large subunit from potato tuber were converted to the homologous catalytic subunit residues, Arg33 and Lys43. The activity of the large subunit mutants cannot be readily tested with a co-expressed wild-type small (catalytic) subunit because of the intrinsic activity of the latter. We co-expressed the regulatory-subunit mutants with SmallD145N, an inactive S subunit in which the catalytic Asp145 was mutated. The activity of the small (catalytic) subunit was reduced more than three orders of magnitude. Coexpression of the L subunit double mutant LargeK44R/T54K with SmallD145N generated an enzyme with considerable activity, 10% and 18% of the wildtype enzyme, in the ADP-glucose synthetic and pyrophosphorolytic direction, respectively. Replacement of those two residues in the small subunit by the homologous amino acids in the L subunits (mutations R33K and K43T) decreased the activity one and two orders of magnitude. The wild-type enzyme and SmallD145NLargeK44R/T54K had very similar kinetic properties indicating that the substrate site has been conserved. The fact that only two mutations in the L subunit restored enzyme activity is very strong evidence that the large subunit is derived from the catalytic ancestor. Previous results showed that Asp145 in the small subunit of the wild-type is essential for catalysis, whereas the homologous Asp160 in the Large WT subunit is not. However, in this study, mutation D160N or D160E in the LK44R/T54K subunit abolished the activity, which shows the ancestral essential role of this residue and confirms that the catalysis of SmallD145NLarge K44R/T54K occurs in the L(b) subunit. A phylogenetic tree of the ADP-Glc PPases present in photosynthetic eukaryotes also sheds information about the origin of the subunits. The tree showed that plant Small and Large subunits can be divided into two and four distinct groups, respectively. The two main groups of S subunits are from dicot and monocot plants, whereas Large subunit groups correlate better with their documented tissue expression. The first Large-subunit group is generally expressed in photosynthetic tissues and comprises Large subunits from dicots and monocots. Group II displays a broader expression pattern, whereas groups III and IV are expressed in storage organs (roots, stems, tubers, seeds). Subunits from group III are only from dicot plants, whereas group IV are seed-specific subunits from monocots. These last two groups stem from the same branch of the phylogenetic tree and split before monocot and dicot separation. Thus few as two mutations turned the L subunit from Solanum tuberosum catalytic, showing that L and S subunits share a common catalytic ancestor, rather than a non-catalytic one. The L subunit evolved to have a regulatory role, lost catalytic residues more than 130 million years ago before monocots and dicots diverged, and preserved, possibly as a byproduct, the active site domain.« less

Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex.

PubMed

Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R; Elbing, Karin; Schmidt, Martin C

2010-01-01

Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

Carney Complex: an update

PubMed Central

Correa, Ricardo; Salpea, Paraskevi; Stratakis, Constantine

2015-01-01

Carney Complex (CNC) is a rare autosomal dominant syndrome, characterized by pigmented lesions of the skin and mucosa, cardiac, cutaneous and other myxomas, and multiple endocrine tumors. The disease is caused by inactivating mutations or large deletions of the PRKAR1A gene located at 17q22–24 coding for the regulatory subunit type I alpha of protein kinase A (PKA) gene. Most recently, components of the complex have been associated with defects of other PKA subunits, such as the catalytic subunits PRKACA (adrenal hyperplasia) and PRKACB (pigmented spots, myxomas, pituitary adenomas). In this report, we review CNC, its clinical features, diagnosis, treatment, and molecular etiology including PRKAR1A mutations and the newest on PRKACA and PRKACB defects especially as they pertain to adrenal tumors and Cushing’s syndrome. PMID:26130139

Relative changes in the abundance of branchial Na(+)/K(+)-ATPase alpha-isoform-like proteins in marine euryhaline milkfish (Chanos chanos) acclimated to environments of different salinities.

PubMed

Tang, Cheng-Hao; Chiu, Yu-Huei; Tsai, Shu-Chuan; Lee, Tsung-Han

2009-08-01

Previous studies revealed that upon salinity challenge, milkfish (Chanos chanos), the euryhaline teleost, exhibited adaptive changes in branchial Na(+)/K(+)-ATPase (NKA) activity with different Na(+) and K(+) affinities. Since alteration of activity and ion-affinity may be influenced by changes in different isoforms of NKA alpha-subunit (i.e., the catalytic subunit), it is, thus, intriguing to compare the patterns of protein abundance of three major NKA alpha-isoform-like proteins (i.e., alpha1, alpha2, and alpha3) in the gills of euryhaline milkfish following salinity challenge. The protein abundance of three NKA alpha-isoform-like proteins in gills of milkfish reared in seawater (SW), fresh water (FW), as well as hypersaline water (HSW, 60 per thousand) were analyzed by immunoblotting. In the acclimation experiments, the SW group revealed significantly higher levels of NKA alpha1- and alpha3-like proteins than the FW or HSW group. Time-course experiments on milkfish that were transferred from SW to HSW revealed the abundance of branchial NKA alpha1-like and alpha3-like proteins decreased significantly after 96 and 12 hr, respectively, and no significant difference was found in NKA alpha2-like protein. Furthermore, when fish were transferred from SW to FW, the amounts of NKA alpha1- and alpha3-like proteins was significantly decreased after 96 hr. Taken together, acute and chronic changes in the abundance of branchial NKA alpha1- and alpha3-like proteins may fulfill the requirements of altering NKA activity with different Na(+) or K(+) affinity for euryhaline milkfish acclimated to environments of various salinities. 2009 Wiley-Liss, Inc.

NMR Studies of the C-Terminus of alpha4 Reveal Possible Mechanism of Its Interaction with MID1 and Protein Phosphatase 2A

PubMed Central

Du, Haijuan; Massiah, Michael A.

2011-01-01

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1. PMID:22194938

An Fe-S cluster in the conserved Cys-rich region in the catalytic subunit of FAD-dependent dehydrogenase complexes.

PubMed

Shiota, Masaki; Yamazaki, Tomohiko; Yoshimatsu, Keiichi; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2016-12-01

Several bacterial flavin adenine dinucleotide (FAD)-harboring dehydrogenase complexes comprise three distinct subunits: a catalytic subunit with FAD, a cytochrome c subunit containing three hemes, and a small subunit. Owing to the cytochrome c subunit, these dehydrogenase complexes have the potential to transfer electrons directly to an electrode. Despite various electrochemical applications and engineering studies of FAD-dependent dehydrogenase complexes, the intra/inter-molecular electron transfer pathway has not yet been revealed. In this study, we focused on the conserved Cys-rich region in the catalytic subunits using the catalytic subunit of FAD dependent glucose dehydrogenase complex (FADGDH) as a model, and site-directed mutagenesis and electron paramagnetic resonance (EPR) were performed. By co-expressing a hitch-hiker protein (γ-subunit) and a catalytic subunit (α-subunit), FADGDH γα complexes were prepared, and the properties of the catalytic subunit of both wild type and mutant FADGDHs were investigated. Substitution of the conserved Cys residues with Ser resulted in the loss of dye-mediated glucose dehydrogenase activity. ICP-AEM and EPR analyses of the wild-type FADGDH catalytic subunit revealed the presence of a 3Fe-4S-type iron-sulfur cluster, whereas none of the Ser-substituted mutants showed the EPR spectrum characteristic for this cluster. The results suggested that three Cys residues in the Cys-rich region constitute an iron-sulfur cluster that may play an important role in the electron transfer from FAD (intra-molecular) to the multi-heme cytochrome c subunit (inter-molecular) electron transfer pathway. These features appear to be conserved in the other three-subunit dehydrogenases having an FAD cofactor. Copyright © 2016 Elsevier B.V. All rights reserved.

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha

PubMed Central

Senior, Alan E.; Muharemagi, Alma; Wilke-Mounts, Susan

2008-01-01

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, competent in nucleotide binding, and had normal N-terminal sequence. In F1-subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiment showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming, and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector. PMID:17176112

Class IA phosphoinositide 3-kinase regulates heart size and physiological cardiac hypertrophy.

PubMed

Luo, Ji; McMullen, Julie R; Sobkiw, Cassandra L; Zhang, Li; Dorfman, Adam L; Sherwood, Megan C; Logsdon, M Nicole; Horner, James W; DePinho, Ronald A; Izumo, Seigo; Cantley, Lewis C

2005-11-01

Class I(A) phosphoinositide 3-kinases (PI3Ks) are activated by growth factor receptors, and they regulate, among other processes, cell growth and organ size. Studies using transgenic mice overexpressing constitutively active and dominant negative forms of the p110alpha catalytic subunit of class I(A) PI3K have implicated the role of this enzyme in regulating heart size and physiological cardiac hypertrophy. To further understand the role of class I(A) PI3K in controlling heart growth and to circumvent potential complications from the overexpression of dominant negative and constitutively active proteins, we generated mice with muscle-specific deletion of the p85alpha regulatory subunit and germ line deletion of the p85beta regulatory subunit of class I(A) PI3K. Here we show that mice with cardiac deletion of both p85 subunits exhibit attenuated Akt signaling in the heart, reduced heart size, and altered cardiac gene expression. Furthermore, exercise-induced cardiac hypertrophy is also attenuated in the p85 knockout hearts. Despite such defects in postnatal developmental growth and physiological hypertrophy, the p85 knockout hearts exhibit normal contractility and myocardial histology. Our results therefore provide strong genetic evidence that class I(A) PI3Ks are critical regulators for the developmental growth and physiological hypertrophy of the heart.

YC-1 BINDING TO THE BETA SUBUNIT OF SOLUBLE GUANYLYL CYCLASE OVERCOMES ALLOSTERIC INHIBITION BY THE ALPHA SUBUNIT

PubMed Central

Purohit, Rahul; Fritz, Bradley G.; The, Juliana; Issaian, Aaron; Weichsel, Andrzej; David, Cynthia L.; Campbell, Eric; Hausrath, Andrew C.; Rassouli-Taylor, Leida; Garcin, Elsa D.; Gage, Matthew J.; Montfort, William R.

2014-01-01

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity. PMID:24328155

Multiple alpha subunits of integrin are involved in cell-mediated responses of the Manduca immune system.

PubMed

Zhuang, Shufei; Kelo, Lisha; Nardi, James B; Kanost, Michael R

2008-01-01

The cell-mediated responses of the insect innate immune system-phagocytosis, nodulation, encapsulation-involve multiple cell adhesion molecules of hemocyte surfaces. A hemocyte-specific (HS) integrin and a member of the immunoglobulin (Ig) superfamily (neuroglian) are involved in the encapsulation response of hemocytes in Manduca sexta. In addition, two new integrin alpha (alpha) subunits have been found on these hemocytes. The alpha2 subunit is mainly expressed in epidermis and Malphigian tubules, whereas the alpha3 subunit is primarily expressed on hemocytes and fat body cells. Of the three known alpha subunits, the alpha1 subunit found in HS integrin is the predominant subunit of hemocytes. Cell adhesion assays indicate that alpha2 belongs to the integrin family with RGD-binding motifs, confirming the phylogenetic analysis of alpha subunits based on the amino-acid sequence alignment of different alpha subunits. Double-stranded RNAs (dsRNAs) targeting each of these three integrin alpha subunits not only specifically decreased transcript expression of each alpha subunit in hemocytes, but also abolished the cell-mediated encapsulation response of hemocytes to foreign surfaces. The individual alpha subunits of M. sexta integrins, like their integrin counterparts in mammalian immune systems, have critical, individual roles in cell-substrate and cell-cell interactions during immune responses.

Overexpression of PP2A-C5 that encodes the catalytic subunit 5 of protein phosphatase 2A in Arabidopsis confers better root and shoot development under salt conditions

USDA-ARS?s Scientific Manuscript database

Protein phosphatase 2A (PP2A) is an enzyme consisting of three subunits: a scaffolding A subunit, a regulatory B subunit and a catalytic C subunit. PP2As were shown to play diverse roles in eukaryotes. In this study, the function of the Arabidopsis PP2A-C5 gene that encodes the catalytic subunit 5 o...

Structure and function of PA4872 from Pseudomonas aeruginosa, a novel class of oxaloacetate decarboxylase from the PEP mutase/isocitrate lyase superfamily.

PubMed

Narayanan, Buvaneswari C; Niu, Weiling; Han, Ying; Zou, Jiwen; Mariano, Patrick S; Dunaway-Mariano, Debra; Herzberg, Osnat

2008-01-08

Pseudomonas aeruginosa PA4872 was identified by sequence analysis as a structurally and functionally novel member of the PEP mutase/isocitrate lyase superfamily and therefore targeted for investigation. Substrate screens ruled out overlap with known catalytic functions of superfamily members. The crystal structure of PA4872 in complex with oxalate (a stable analogue of the shared family alpha-oxyanion carboxylate intermediate/transition state) and Mg2+ was determined at 1.9 A resolution. As with other PEP mutase/isocitrate lyase superfamily members, the protein assembles into a dimer of dimers with each subunit adopting an alpha/beta barrel fold and two subunits swapping their barrel's C-terminal alpha-helices. Mg2+ and oxalate bind in the same manner as observed with other superfamily members. The active site gating loop, known to play a catalytic role in the PEP mutase and lyase branches of the superfamily, adopts an open conformation. The Nepsilon of His235, an invariant residue in the PA4872 sequence family, is oriented toward a C(2) oxygen of oxalate analogous to the C(3) of a pyruvyl moiety. Deuterium exchange into alpha-oxocarboxylate-containing compounds was confirmed by 1H NMR spectroscopy. Having ruled out known activities, the involvement of a pyruvate enolate intermediate suggested a decarboxylase activity of an alpha-oxocarboxylate substrate. Enzymatic assays led to the discovery that PA4872 decarboxylates oxaloacetate (kcat = 7500 s(-1) and Km = 2.2 mM) and 3-methyloxaloacetate (kcat = 250 s(-1) and Km = 0.63 mM). Genome context of the fourteen sequence family members indicates that the enzyme is used by select group of Gram-negative bacteria to maintain cellular concentrations of bicarbonate and pyruvate; however the decarboxylation activity cannot be attributed to a pathway common to the various bacterial species.

The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

PubMed

Asher, O; Jensen, B S; Lupu-Meiri, M; Oron, Y; Fuchs, S

1998-04-17

The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

Carcinogenesis of PIK3CA

PubMed Central

2013-01-01

PIK3CA is the most frequently mutated oncogene in human cancers. PIK3CA is phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha. It controls cell growth, proliferation, motility, survival, differentiation and intracellular trafficking. In most of human cancer alteration occurred frequently in the alpha isoform of phosphatidylinositol 3 kinase. PIK3CA mutations were most frequent in endometrial, ovarian, colorectal, breast, cervical, squamous cell cancer of the head and neck, chondroma, thyroid carcinoma and in cancer family syndrome. Inhibition of PI3K signaling can diminish cell proliferation, and in some circumstances, promote cell death. Consequently, components of this pathway present attractive targets for cancer therapeutics. A number of PI3K pathway inhibitors have been developed and used. PI3K inhibitors (both pan-PI3K and isoform-specific PI3K inhibitors), dual PI3K-mTOR inhibitors that are catalytic site inhibitors of the p110 isoforms and mTOR (the kinase component of both mTORC1 and mTORC2), mTOR catalytic site inhibitors, and AKT inhibitors are the most advanced in the clinic. They are approved for the treatment of several carcinomas. PMID:23768168

Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

NASA Astrophysics Data System (ADS)

Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T. V.; Alyethodi, Rafeeque R.; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B.

2015-12-01

Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly ( P < 0.05) higher during the thermal stress. Pearson correlation coefficient analysis revealed that the expression of ATPase Β1, ATPase B2, and ATPase B3 is highly correlated ( P < 0.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

Crystal Structure of the 25 kDa Subunit of Human Cleavage Factor I{m}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coseno,M.; Martin, G.; Berger, C.

Cleavage factor Im is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein-protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic {alpha}/{beta}/{alpha} fold and a conserved catalytic sequence or Nudix box. We present heremore » the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Angstroms, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3'-end processing.« less

Purification and characterization of protein phosphatase 2A from petals of the tulip Tulipa gesnerina.

PubMed

Azad, Md Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

2006-11-30

The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748- fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.

Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits.

PubMed

Urban, C; Salton, M R

1983-08-31

The F1-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F1S, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230-240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F1-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F1 are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual delta- and epsilon-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F1; and (4) all subunit antibodies as well as anti-native F1 were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F1 and anti-alpha- and anti-beta-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F1-ATPases and in general may offer an additional method for the examination of multimeric proteins.

Identification of functional domains within the alpha and beta subunits of beta-hexosaminidase A through the expression of alpha-beta fusion proteins.

PubMed

Tse, R; Wu, Y J; Vavougios, G; Hou, Y; Hinek, A; Mahuran, D J

1996-08-20

There are three human beta-hexosaminidase isozymes which are composed of all possible dimeric combinations of an alpha and/or a beta subunit; A (alpha beta), and B (beta beta), and S (alpha alpha). The amino acid sequences of the two subunits are 60% identical. The homology between the two chains varies with the middle > the carboxy-terminal > > the amino-terminal portions. Although dimerization is required for activity, each subunit contains its own active site and differs in its substrate specificity and thermal stability. The presence of the beta subunit in hexosaminidase A also influences the substrate specificity of the alpha subunit; e.g., in vivo only the A heterodimer can hydrolyze GM2 ganglioside. In this report, we localize functional regions in the two subunits by cellular expression of alpha/beta fusion proteins joined at adjacently aligned residues. First, a chimeric alpha/beta chain was made by replacing the least well-conserved amino-terminal section of the beta chain with the corresponding alpha section. The biochemical characteristics of this protein were nearly identical to hexosaminidase B. Therefore, the most dissimilar regions in the subunits are not responsible for their dissimilar biochemical properties. A second fusion protein was made that also included the more homologous middle section of the alpha chain. This protein expressed the substrate specificity unique to isozymes containing an alpha subunit (A and S). We conclude that the region responsible for the ability of the alpha subunit to bind negatively charged substrates is located within residues alpha 132-283. Interestingly, the remaining carboxy-terminal section from the beta chain, beta 316-556, was sufficient to allow this chimera to hydrolyze GM2 ganglioside with 10% the specific activity of heterodimeric hexosaminidase A. Thus, the carboxy-terminal section of each subunit is likely involved in subunit-subunit interactions.

Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

NASA Technical Reports Server (NTRS)

Li, H.; Roux, S. J.

1992-01-01

Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

Structure and Function of PA4872 from Pseudomonas aeruginosa, a Novel Class of Oxaloacetate Decarboxylase from the PEP Mutase/Isocitrate Lyase Superfamily

DOE Office of Scientific and Technical Information (OSTI.GOV)

Narayanan, Buvaneswari C.; Niu, Weiling; Han, Ying

2008-06-30

Pseudomonas aeruginosa PA4872 was identified by sequence analysis as a structurally and functionally novel member of the PEP mutase/isocitrate lyase superfamily and therefore targeted for investigation. Substrate screens ruled out overlap with known catalytic functions of superfamily members. The crystal structure of PA4872 in complex with oxalate (a stable analogue of the shared family R-oxyanion carboxylate intermediate/transition state) and Mg{sup 2+} was determined at 1.9 {angstrom} resolution. As with other PEP mutase/isocitrate lyase superfamily members, the protein assembles into a dimer of dimers with each subunit adopting an {alpha}/{beta} barrel fold and two subunits swapping their barrel's C-terminal {alpha}-helices. Mg2+more » and oxalate bind in the same manner as observed with other superfamily members. The active site gating loop, known to play a catalytic role in the PEP mutase and lyase branches of the superfamily, adopts an open conformation. The N{sup {epsilon}} of His235, an invariant residue in the PA4872 sequence family, is oriented toward a C(2) oxygen of oxalate analogous to the C(3) of a pyruvyl moiety. Deuterium exchange into {alpha}-oxocarboxylate-containing compounds was confirmed by {sup 1}H NMR spectroscopy. Having ruled out known activities, the involvement of a pyruvate enolate intermediate suggested a decarboxylase activity of an {alpha}-oxocarboxylate substrate. Enzymatic assays led to the discovery that PA4872 decarboxylates oxaloacetate (k{sub cat}) = 7500 s{sup -1} and K{sub m} = 2.2 mM) and 3-methyloxaloacetate (k{sub cat}) = 250 s{sup -1} and K{sub m} = 0.63 mM). Genome context of the fourteen sequence family members indicates that the enzyme is used by select group of Gram-negative bacteria to maintain cellular concentrations of bicarbonate and pyruvate; however the decarboxylation activity cannot be attributed to a pathway common to the various bacterial species.« less

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

PubMed

Cheng, C; Prince, L S; Snyder, P M; Welsh, M J

1998-08-28

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

Nicotinic acetylcholine receptor alpha5 subunits modulate oxotremorine-induced salivation and tremor.

PubMed

Wang, Ningshan; Orr-Urtreger, Avi; Chapman, Joab; Rabinowitz, Ruth; Korczyn, Amos D

2004-07-15

Neuronal nicotinic acetylcholine receptors (nAChRs) are composed of 12 subunits (alpha2-alpha10 and beta2-beta4). alpha5 Subunits, expressed throughout the central nervous system (CNS) and the autonomic nervous system (ANS), possess unique pharmacological properties. The effects of oxotremorine (OXO) on autonomic functions and tremor were examined in mice lacking alpha5 nAChR subunits (alpha5-/-) and compared with those in wild-type (WT) control mice. The alpha5-/- mice showed significantly increased salivation and tremor responses to OXO. The hypothermia, bradycardia and defecation induced by OXO were of similar magnitudes in the two mouse strains. The enhanced OXO effects in alpha5-/- mice indicate inhibitory effects of alpha5 subunits in autonomic ganglia, and support the participation of these subunits in cholinergic transmission in autonomic ganglia.

Structure of the protein phosphatase 2A holoenzyme.

PubMed

Xu, Yanhui; Xing, Yongna; Chen, Yu; Chao, Yang; Lin, Zheng; Fan, Eugene; Yu, Jong W; Strack, Stefan; Jeffrey, Philip D; Shi, Yigong

2006-12-15

Protein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B'/B56/PR61. Surprisingly, the B'/PR61 subunit has a HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B'/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B'/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A.

Function of Several Critical Amino Acids in Human Pyruvate Dehydrogenase Revealed by Its Structure

NASA Technical Reports Server (NTRS)

Korotchkina, Lioubov G.; Ciszak, E.; Patel, M.

2004-01-01

Pyruvate dehydrogenase (E1), an alpha 2 beta 2 tetramer, catalyzes the oxidative decarboxylation of pyruvate and reductive acetylation of lipoyl moieties of the dihydrolipoamide acetyltransferase. The roles of beta W135, alpha P188, alpha M181, alpha H15 and alpha R349 of E1 determined by kinetic analysis were reassessed by analyzing the three-dimensional structure of human E1. The residues identified above are found to play a structural role rather than being directly involved in catalysis: beta W135 is the center residue in the hydrophobic interaction between beta and beta' subunits; alpha P188 and alpha M181 are critical for the conformation of the TPP-binding motif and interaction between alpha and beta subunits; alpha H15, is necessary for the organization of the N-terminus of alpha and alpha'; subunits and alpha R349 supports the interaction of the C-terminus of the alpha subunits with the beta subunits. Analysis of several critical E1 residues confirms the importance of residues distant from the active site for subunit interactions and enzyme function.

The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

PubMed

Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

1997-10-01

The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon terminals. The type 2 neurons comprised 20% of the total population of alpha1-subunit-immunoreactive neurons. The remaining large-sized alpha1-immunoreactive cells were designated type 3 cells; they were positive for parvalbumin and were distinguished by long branching dendrites extending dorsally for 600-800 microm into the striatum. These neurons comprised 5% of the total population of alpha1-subunit-immunoreactive neurons and were surrounded by enkephalin-immunoreactive terminals. Electron microscopy showed that the alpha1-subunit type 3 neurons had an indented nuclear membrane and were densely covered with small axon terminals which established alpha1-subunit-immunoreactive symmetrical synaptic contacts with the soma and dendrites. These results provide a detailed characterization of the distribution, morphology and chemical characteristics of the alpha1-subunit-immunoreactive neurons in the rat striatum and suggest that the type 1 and type 2 neurons comprise of separate populations of striatal interneurons while the type 3 neurons may represent the large striatonigral projection neurons described by Bolam et al. [Bolam J. P., Somogyi P., Totterdell S. and Smith A. D. (1981) Neuroscience 6, 2141-2157.].

The Crystal Structure of the Ring-Hydroxylating Dioxygenase from Sphingomonas CHY-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jakoncic,J.; Jouanneau, Y.; Meyer, C.

The ring-hydroxylating dioxygenase (RHD) from Sphingomonas CHY-1 is remarkable due to its ability to initiate the oxidation of a wide range of polycyclic aromatic hydrocarbons (PAHs), including PAHs containing four- and five-fused rings, known pollutants for their toxic nature. Although the terminal oxygenase from CHY-1 exhibits limited sequence similarity with well characterized RHDs from the naphthalene dioxygenase family, the crystal structure determined to 1.85 {angstrom} by molecular replacement revealed the enzyme to share the same global {alpha}{sub 3}{beta}{sub 3} structural pattern. The catalytic domain distinguishes itself from other bacterial non-heme Rieske iron oxygenases by a substantially larger hydrophobic substrate bindingmore » pocket, the largest ever reported for this type of enzyme. While residues in the proximal region close to the mononuclear iron atom are conserved, the central region of the catalytic pocket is shaped mainly by the side chains of three amino acids, Phe350, Phe404 and Leu356, which contribute to the rather uniform trapezoidal shape of the pocket. Two flexible loops, LI and LII, exposed to the solvent seem to control the substrate access to the catalytic pocket and control the pocket length. Compared with other naphthalene dioxygenases residues Leu223 and Leu226, on loop LI, are moved towards the solvent, thus elongating the catalytic pocket by at least 2 {angstrom}. An 11 {angstrom} long water channel extends from the interface between the {alpha} and {beta} subunits to the catalytic site. The comparison of these structures with other known oxygenases suggests that the broad substrate specificity presented by the CHY-1 oxygenase is primarily due to the large size and particular topology of its catalytic pocket and provided the basis for the study of its reaction mechanism.« less

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

PubMed

Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

Alteration of GABAergic synapses and gephyrin clusters in the thalamic reticular nucleus of GABAA receptor alpha3 subunit-null mice.

PubMed

Studer, Remo; von Boehmer, Lotta; Haenggi, Tatjana; Schweizer, Claude; Benke, Dietmar; Rudolph, Uwe; Fritschy, Jean-Marc

2006-09-01

Multiple GABAA-receptor subtypes are assembled from alpha, beta and gamma subunit variants. GABAA receptors containing the alpha3 subunit represent a minor population with a restricted distribution in the CNS. In addition, they predominate in monoaminergic neurons and in the nucleus reticularis thalami (nRT), suggesting a role in the regulation of cortical function and sleep. Mice with a targeted deletion of the alpha3 subunit gene (alpha3(0/0)) are viable and exhibit a subtle behavioural phenotype possibly related to dopaminergic hyperfunction. Here, we investigated immunohistochemically the consequences of the loss of alpha3 subunit for maturation of GABAA receptors and formation of GABAergic synapses in the nRT. Throughout postnatal development, the regional distribution of the alpha1, alpha2, or alpha5 subunit was unaltered in alpha3(0/0) mice and the prominent alpha3 subunit staining of nRT neurons in wildtype mice was not replaced. Subcellularly, as seen by double immunofluorescence, the alpha3 and gamma2 subunit were clustered at postsynaptic sites in the nRT of adult wildtype mice along with the scaffolding protein gephyrin. In alpha3(0/0) mice, gamma2 subunit clustering was disrupted and gephyrin formed large aggregates localized at the cell surface, but unrelated to postsynaptic sites, indicating that nRT neurons lack postsynaptic GABAA receptors in mutant mice. Furthermore, GABAergic terminals were enlarged and reduced in number, suggesting a partial deficit of GABAergic synapses. Therefore, GABAA receptors are required for gephyrin clustering and long-term synapse maintenance. The absence of GABAA-mediated transmission in the nRT may have a significant impact on the function of the thalamo-cortical loop of alpha3(0/0) mice.

Effects of troxerutin on cognitive deficits and glutamate cysteine ligase subunits in the hippocampus of streptozotocin-induced type 1 diabetes mellitus rats.

PubMed

Zhang, Songyun; Li, Hongyan; Zhang, Lihui; Li, Jie; Wang, Ruiying; Wang, Mian

2017-02-15

Increasing evidence demonstrates an association between diabetes and hippocampal neuron damage. This study aimed to determine the effects of troxerutin on cognitive deficits and glutamate cysteine ligase subunits (GCLM and GCLC) in the hippocampus of streptozotocin-induced type 1 diabetes mellitus (T1DM) rats. At 12weeks after streptozotocin injection, T1DM rats were randomly divided into 4 groups (n=15 each group) to receive no treatment (T1DM), saline (T1DM+saline), alpha-lipoic acid (T1DM+alpha-lipoic acid), and troxerutin (T1DM+troxerutin), respectively, for 6weeks. Meanwhile, 10 control animals (NC group) were assessed in parallel. Learning performance was evaluated by the Morris water maze. After treatment, hippocampi were collected for pathological examination by hematoxylin and eosin (H&E) staining. Next, hippocampal superoxide dismutase (SOD) activity, and malondialdehyde (MDA) and glutathione (GSH) levels were assessed. Finally, glutamate cysteine ligase catalytic (GCLC) and glutamate cysteine ligase modifier (GCLM) subunit mRNA and protein levels were quantified by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. Compared with T1DM and T1DM+saline groups, escape latency was overtly reduced in T1DM+alpha-lipoic acid and T1DM+troxerutin groups. Significantly increased GCLM and GCLC mRNA levels, GCLC protein amounts, SOD activity, and GSH levels, and reduced MDA amounts were observed in T1DM+alpha-lipoic acid and T1DM+troxerutin groups. In T1DM and T1DM+saline groups, H&E staining showed less pyramidal cells in the hippocampus, with disorganized layers, karyopyknosis, decreased endochylema, and cavitation, effects relieved in T1DM+alpha-lipoic acid and T1DM+troxerutin groups. Troxerutin alleviates oxidative stress and promotes learning in streptozotocin-induced T1DM rats, a process involving GCLC expression. Copyright © 2016 Elsevier B.V. All rights reserved.

Epoxyethylglycyl peptides as inhibitors of oligosaccharyltransferase: double-labelling of the active site.

PubMed

Bause, E; Wesemann, M; Bartoschek, A; Breuer, W

1997-02-15

Pig liver oligosaccharyltransferase (OST) is inactivated irreversibly by a hexapeptide in which threonine has been substituted by epoxyethylglycine in the Asn-Xaa-Thr glycosylation triplet. Incubation of the enzyme in the presence of Dol-PP-linked [14C]oligosaccharides and the N-3,5-dinitrobenzoylated epoxy derivative leads to the double-labelling of two subunits (48 and 66 kDa) of the oligomeric OST complex, both of which are involved in the catalytic activity. Labelling of both subunits was blocked competitively by the acceptor peptide N-benzoyl-Asu-Gly-Thr-NHCH3 and by the OST inhibitor N-benzoyl-alpha,gamma-diaminobutyric acid-Gly-Thr-NHCH3, but not by an analogue derived from the epoxy-inhibitor by replacing asparagine with glutamine. Our data clearly show that double-labelling is an active-site-directed modification, involving inhibitor glycosylation at asparagine and covalent attachment of the glycosylated inhibitor, via the epoxy group, to the enzyme. Double-labelling of OST can occur as the result of either a consecutive or a syn-catalytic reaction sequence. The latter mechanism, during the course of which OST catalyses its own 'suicide' inactivation, is more likely, as suggested by indirect experimental evidence. The syn-catalytic mechanism corresponds with our current view of the functional role of the acceptor site Thr/Ser acting as a hydrogen-bond acceptor, not a donor, during transglycosylation.

Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, T.; Weintraub, B.D.

1985-04-01

The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing (/sup 14/C)alanine and (/sup 3/H) glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, (/supmore » 14/C)alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. (/sup 3/H)Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function.« less

The eukaryotic RNA exosome: same scaffold but variable catalytic subunits.

PubMed

Lykke-Andersen, Søren; Tomecki, Rafal; Jensen, Torben Heick; Dziembowski, Andrzej

2011-01-01

The RNA exosome is a versatile ribonucleolytic protein complex that participates in a multitude of cellular RNA processing and degradation events. It consists of an invariable nine-subunit core that associates with a variety of enzymatically active subunits and co-factors. These contribute to or even provide the catalytic activity and substrate specificity of the complex. The S. cerevisiae exosome has been intensively studied since its discovery in 1997 and thus serves as the archetype of eukaryotic exosomes. Notably, its catalytic potential, derived exclusively from associated subunits, differs between the nuclear and cytoplasmic versions of the complex. The same holds true for other eukaryotes, however, recent discoveries from various laboratories including our own have revealed that there are variations on this theme. Here, we review the latest findings concerning catalytic subunits of eukaryotic exosomes, and we discuss the apparent need for differential composition and subcellular distribution of exosome variants.

Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

NASA Astrophysics Data System (ADS)

Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

1995-02-01

The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

Purification and characterization of the glycoprotein hormone. cap alpha. -subunit-like material secreted by HeLa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cox, G.S.; Rimerman, R.A.

1988-08-23

The protein secreted by HeLa cells that cross-reacts with antiserum developed against the ..cap alpha..-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10/sup 5/ ng of ..cap alpha../mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-..cap alpha.. had a composition very similar to that of the urinary hCG ..cap alpha..-subunit. However, comparison of hCG-..cap alpha.. and HeLa-..capmore » alpha.. demonstrated that the tumor-associated subunit was not identical with its normal counterpart. The purified tumor protein had an apparent molecular weight greater than that of the urinary ..cap alpha..-subunit when analyzed by SDS-PAGE, and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI. Immunoprecipitation and electrophoresis of ..cap alpha..-subunit from HeLa cultures labeled with (/sup 3/H)fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-..cap alpha.. hydrosylates by HPLC confirmed previous reports that the placental subunit does not contain fucose. The results indicate that, regardless of whether or not a single ..cap alpha..-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors.« less

Cellulose microfibril structure: inspirations from plant diversity

NASA Astrophysics Data System (ADS)

Roberts, A. W.

2018-03-01

Cellulose microfibrils are synthesized at the plasma membrane by cellulose synthase catalytic subunits that associate to form cellulose synthesis complexes. Variation in the organization of these complexes underlies the variation in cellulose microfibril structure among diverse organisms. However, little is known about how the catalytic subunits interact to form complexes with different morphologies. We are using an evolutionary approach to investigate the roles of different catalytic subunit isoforms in organisms that have rosette-type cellulose synthesis complexes.

Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes.

PubMed

Peng, X; Katz, M; Gerzanich, V; Anand, R; Lindstrom, J

1994-03-01

The alpha-bungarotoxin-binding acetylcholine receptors from the human neuroblastoma cell line SH-SY5Y were found to cross-react with some monoclonal antibodies to alpha 7 subunits of nicotinic acetylcholine receptors from chicken brain. The human alpha 7 subunit cDNA from SH-SY5Y was cloned, revealing 94% amino acid sequence identity to rat alpha 7 subunits and 92% identity to chicken alpha 7 subunits. Native human alpha 7 receptors showed affinities for some ligands similar to those previously observed with native chicken alpha 7 receptors, but for other ligands there were large species-specific differences in binding affinity. These results paralleled properties of alpha 7 homomers expressed in Xenopus oocytes. Human alpha 7 homomers exhibited rapidly desensitizing, inwardly rectifying, agonist-induced, cation currents that triggered Ca(2+)-sensitive Cl- channels in the oocytes. A change in efficacy from partial agonist for chicken alpha 7 homomers to full agonist for human alpha 7 homomers was exhibited by 1,1-dimethyl-4-phenylpiperazinium. This result reveals a large species-specific pharmacological difference, despite small differences in alpha 7 sequences. This is important for understanding the effects of these drugs in humans and for identifying amino acids that may contribute to the acetylcholine binding site, for analysis by in vitro mutagenesis. These results also characterize properties of native alpha 7 receptors and alpha 7 homomers that will provide criteria for functional properties expected of structural subunits, when these can be identified, cloned, and coexpressed with alpha 7 subunits.

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

PubMed

Stoyanov, B; Volinia, S; Hanck, T; Rubio, I; Loubtchenkov, M; Malek, D; Stoyanova, S; Vanhaesebroeck, B; Dhand, R; Nürnberg, B

1995-08-04

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

Specific antibodies against Go isoforms reveal the early expression of the Go2 alpha subunit and appearance of Go1 alpha during neuronal differentiation.

PubMed

Rouot, B; Charpentier, N; Chabbert, C; Carrette, J; Zumbihl, R; Bockaert, J; Homburger, V

1992-02-01

We have previously identified two isoforms of Go alpha in membranes of N1E-115 neuroblastoma cells, using an antibody raised against the purified Go alpha subunit; one isoform of the Go alpha subunit (pI 5.80) is present in undifferentiated cells, whereas a more acidic isoform (pI 5.55) appears during differentiation [J. Neurochem. 54:1310-1320 (1990)]. Recently, the Go alpha gene has been shown to encode, by alternative splicing, two polypeptides, Go1 alpha and Go2 alpha, which differ only in their carboxyl-terminal part. To determine unambiguously whether the two Go alpha subunits detected in neuroblastoma cells were actually the products of different mRNAs, rabbit polyclonal antibodies were generated against synthetic peptides (amino acids 291-302) of both sequences. Specificity of the two affinity-purified antipeptide antibodies was assessed on Western blots by comparing their immunoreactivities with those of other G alpha antibodies. On a blotted mixture of purified brain guanine nucleotide-binding proteins, the anti-alpha o1 and anti-alpha o2 peptide antibodies only recognized the 39-kDa Go alpha subunit. Furthermore, the immunological recognition of brain membranes from 15-day-old mouse fetuses by antipeptide antibodies could be specifically blocked by addition of the corresponding antigen. When membrane proteins from differentiated neuroblastoma cells and mouse fetus brain were blotted after two-dimensional gel electrophoresis, the anti-alpha o1 and anti-alpha o2 peptide antibodies labeled a 39-kDa subunit focused at a pI value of 5.55 or 5.80, respectively. Study of the ontogenesis of both Go alpha subunits revealed the predominance of Go2 alpha in the frontal cortex at day 15 of gestation. Thereafter, there was a progressive decline of the Go2 alpha polypeptide to a very low level, concomitant with an increase in the Go1 alpha protein, which plateaued about 15 days after birth to a level 8 times higher than at gestational day 15. Similarly, on neuroblastoma cells, the Go2 alpha subunit was almost exclusively present in undifferentiated cells, and differentiation induced the appearance of the Go1 alpha subunit, with a reduction in the amount of Go2 alpha polypeptide. Thus, the evolution of the two Go alpha subunits during cell differentiation, unambiguously identified with specific antibodies, suggests that neuronal differentiation is responsible for the on/off switch of the expression of the Go alpha isoforms and indicates that Go1 alpha, rather than Go2 alpha, is involved in neurotransmission.

A dynamic alpha-beta inter-subunit agonist signaling complex is a novel feedback mechanism for regulating L-type Ca2+ channel opening.

PubMed

Zhang, Rong; Dzhura, Igor; Grueter, Chad E; Thiel, William; Colbran, Roger J; Anderson, Mark E

2005-09-01

L-type Ca2+ channels are macromolecular protein complexes in neurons and myocytes that open in response to cell membrane depolarization to supply Ca2+ for regulating gene transcription and vesicle secretion and triggering cell contraction. L-type Ca2+ channels include a pore-forming alpha and an auxiliary beta subunit, and alpha subunit openings are regulated by cellular Ca2+ through a mechanism involving the Ca2+-sensing protein calmodulin (CaM) and CaM binding motifs in the alpha subunit cytoplasmic C terminus. Here we show that these CaM binding motifs are "auto-agonists" that increase alpha subunit openings by binding the beta subunit. The CaM binding domains are necessary and sufficient for the alpha subunit C terminus to bind the beta subunit in vitro, and excess CaM blocks this interaction. Addition of CaM binding domains to native cardiac L-type Ca2+ channels in excised cell membrane patches increases openings, and this agonist effect is prevented by excess CaM. Recombinant LTCC openings are also increased by exogenous CaM binding domains by a mechanism requiring the beta subunit, and excess CaM blocks this effect. Thus, the bifunctional ability of the alpha subunit CaM binding motifs to competitively associate with the beta subunit or CaM provides a novel paradigm for feedback control of cellular Ca2+ entry.

Functional and genomic analyses of alpha-solenoid proteins.

PubMed

Fournier, David; Palidwor, Gareth A; Shcherbinin, Sergey; Szengel, Angelika; Schaefer, Martin H; Perez-Iratxeta, Carol; Andrade-Navarro, Miguel A

2013-01-01

Alpha-solenoids are flexible protein structural domains formed by ensembles of alpha-helical repeats (Armadillo and HEAT repeats among others). While homology can be used to detect many of these repeats, some alpha-solenoids have very little sequence homology to proteins of known structure and we expect that many remain undetected. We previously developed a method for detection of alpha-helical repeats based on a neural network trained on a dataset of protein structures. Here we improved the detection algorithm and updated the training dataset using recently solved structures of alpha-solenoids. Unexpectedly, we identified occurrences of alpha-solenoids in solved protein structures that escaped attention, for example within the core of the catalytic subunit of PI3KC. Our results expand the current set of known alpha-solenoids. Application of our tool to the protein universe allowed us to detect their significant enrichment in proteins interacting with many proteins, confirming that alpha-solenoids are generally involved in protein-protein interactions. We then studied the taxonomic distribution of alpha-solenoids to discuss an evolutionary scenario for the emergence of this type of domain, speculating that alpha-solenoids have emerged in multiple taxa in independent events by convergent evolution. We observe a higher rate of alpha-solenoids in eukaryotic genomes and in some prokaryotic families, such as Cyanobacteria and Planctomycetes, which could be associated to increased cellular complexity. The method is available at http://cbdm.mdc-berlin.de/~ard2/.

The PA influenza virus polymerase subunit is a phosphorylated protein.

PubMed

Sanz-Ezquerro, J J; Fernández Santarén, J; Sierra, T; Aragón, T; Ortega, J; Ortín, J; Smith, G L; Nieto, A

1998-03-01

The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by two-dimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [32P]orthophosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine and phosphothreonine epitopes were present in PA, while phosphotyrosine residues were absent, as tested by immunoblotting with specific antibodies. These facts, as well as the presence of multiple consensus sites for casein kinase II (CKII) phosphorylation, prompted us to test the involvement of this kinase in PA covalent modification. PA protein purified by immunoprecipitation could be specifically labelled by the catalytic alpha subunit of human CKII, which was expressed and purified from bacteria. Collectively, these data demonstrate that the PA subunit of the influenza virus RNA polymerase is a phosphoprotein.

Identification and Characterization of an Alternatively Spliced Isoform of the Human Protein Phosphatase 2Aα Catalytic Subunit*

PubMed Central

Migueleti, Deivid L. S.; Smetana, Juliana H. C.; Nunes, Hugo F.; Kobarg, Jörg; Zanchin, Nilson I. T.

2012-01-01

PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation. PMID:22167190

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes.

PubMed

Capmany, G; Mart, M; Santaló, J; Bolton, V N

1998-10-01

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.

A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells.

PubMed

Peters, B P; Krzesicki, R F; Hartle, R J; Perini, F; Ruddon, R W

1984-12-25

Human choriocarcinoma cells (JAR) synthesize the alpha and beta subunits of the glycoprotein hormone chorionic gonadotropin (hCG) (R.W. Ruddon, C.A. Hanson, A. H. Bryan, G.J. Putterman, E.L. White, F. Perini, K. S. Meade, and P.H. Aldenderfer (1980) J. Biol. Chem. 255, 1000-1007). In addition to the hCG dimer (alpha beta), JAR cells secrete uncombined alpha and beta subunits into the culture medium (L.A. Cole, R.J. Hartle, J.A. Laferla, and R.W. Ruddon (1983) Endocrinology 113, 1176-1178). Pulse-chase studies with [35S]methionine or [3H]mannose were carried out in order to compare free alpha, free beta, and the alpha beta dimer with regard to the kinetics of synthesis, N-linked oligosaccharide processing, and secretion and to determine the kinetics of alpha-beta subunit combination. A panel of three antisera was used to immunoprecipitate directly the free subunits and the alpha beta dimer sequentially from the same cell lysates and culture media. The alpha subunit of hCG was synthesized in a slight molar excess (1.2-1.5-fold) over the beta subunit, and alpha beta dimer was rapidly formed by combination of the intracellular alpha and beta precursors. Dimer formation was already apparent in JAR cells following a 10-min biosynthetic labeling incubation with [35S]methionine. The combination of subunits ceased by 30 min of chase even though 51% of alpha and 44% of beta remained free within the cells. Combination of the alpha and beta precursors had occurred before their N-linked oligosaccharides were processed beyond the Man8GlcNAc2 structure. The initial trimming of glucosyl and mannosyl units from the high-mannose oligosaccharides of the hCG precursors occurred more rapidly for free alpha and CG-alpha than for free beta and CG-beta. JAR cells accumulated alpha precursors bearing mostly Man8GlcNAc2 units and beta precursors bearing Man8GlcNAc2 units that represent the substrates of the rate-limiting step in the secretory pathway. In spite of the fact that their N-linked oligosaccharides were trimmed at different rates, free alpha, free beta, and alpha beta dimer were all secreted into the medium at the same rate, with a half-time of 35 min. The secreted hCG forms were stable in the chase medium between 4 and 8h, indicating that extracellular degradation, combination of free subunits to form dimer, or dissociation of dimer to form free subunits did not occur.(ABSTRACT TRUNCATED AT 400 WORDS)

Construction and characterization of the hetero-oligomer of the group II chaperonin from the hyperthermophilic archaeon, Thermococcus sp. strain KS-1.

PubMed

Sahlan, Muhamad; Kanzaki, Taro; Yohda, Masafumi

2009-05-01

The hyperthermophilic archaeon Thermococcus sp. strain KS-1 (T. KS-1) expresses two different chaperonin subunits, alpha and beta, for the folding of its proteins. The composition of the subunits in the hexadecameric double ring changes with temperature. The content of the beta subunit significantly increases according to the increase in temperature. The homo-oligomer of the beta subunit, Cpn beta, is more thermostable than that of the alpha subunit, Cpn alpha. Since Cpn alpha and Cpn beta also have different protein folding activities and interactions with prefoldin, the hetero-oligomer is thought to exhibit different characteristics according to the content of subunits. The hetero-oligomer of the T. KS-1 chaperonin has not been studied, however, because the alpha and beta subunits form hetero-oligomers of varying compositions when they are expressed simultaneously. In this study, we characterized the T. KS-1 chaperonin hetero-oligomer, Cpn alphabeta, containing both alpha and beta in the alternate order, which was constructed by the expression of alpha and beta subunits in a coordinated fashion and protease digestion. Cpn alphabeta protected citrate synthase from thermal aggregation, promoted the folding of acid-denatured GFP in an ATP-dependent manner, and exhibited an ATP-dependent conformational change. The yield of refolded GFP generated by Cpn alphabeta was almost equivalent to that generated by Cpn beta but lower than that generated by Cpn alpha. In contrast, Cpn alphabeta exhibited almost the same level of thermal stability as Cpn alpha, which was lower than that of Cpn beta. The affinity of Cpn alphabeta to prefoldin was found to be between those of Cpn alpha and Cpn beta, as expected.

Crystallization and preliminary X-ray diffraction analysis of the P3 RNA domain of yeast ribonuclease MRP in a complex with RNase P/MRP protein components Pop6 and Pop7.

PubMed

Perederina, Anna; Esakova, Olga; Quan, Chao; Khanova, Elena; Krasilnikov, Andrey S

2010-01-01

Eukaryotic ribonucleases P and MRP are closely related RNA-based enzymes which contain a catalytic RNA component and several protein subunits. The roles of the protein subunits in the structure and function of eukaryotic ribonucleases P and MRP are not clear. Crystals of a complex that included a circularly permuted 46-nucleotide-long P3 domain of the RNA component of Saccharomyces cerevisiae ribonuclease MRP and selenomethionine derivatives of the shared ribonuclease P/MRP protein components Pop6 (18.2 kDa) and Pop7 (15.8 kDa) were obtained using the sitting-drop vapour-diffusion method. The crystals belonged to space group P4(2)22 (unit-cell parameters a = b = 127.2, c = 76.8 A, alpha = beta = gamma = 90 degrees ) and diffracted to 3.25 A resolution.

The electrophoretically 'slow' and 'fast' forms of the alpha 2-macroglobulin molecule.

PubMed Central

Barrett, A J; Brown, M A; Sayers, C A

1979-01-01

alpha 2-Macroglobulin (alpha 2M) was isolated from human plasma by a four-step procedure: poly(ethylene glyco) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No contaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis, and was designated 'S-alpha 2M'. S-alpha 2M bound about 2 mol of trypsin/mol. Treatment of S-alpha 2M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis, and lacking proteinase-binding activity (F-alpha 2M). The electrophoretic mobility of the F-alpha 2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the alpha 2M to a conformation change, but there was no evidence of a change in pI or Strokes radius. Electrophoresis of S-alpha 2M in the presence of sodium dodecylsulphate gave results consistent with the view that the alpha 2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be 'nicked' into two-thires-length and one-third-length chains, however. This was not apparent with F-alpha 2M produced by ammonium salts. F-alpha 2M produced by trypsin showed two new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-alpha 2M gave 'rockets' 12-29% lower than those with S-alpha 2M. The nature of the interactions between subunits in S-alpha 2M and F-alpha 2M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-alpha 2M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-alpha 2M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not show the proteinase-binding activity characteristic of the intact alpha 2M. F-alpha 2M was less easily dissociated than was S-alpha 2M. S-alpha 2M was readily dissociated to the quarter-subunits by mild reduction, with the formation of 3-4 new thiol groups per subunit. Inact reactive alpha 2M could then be regenerated in high yield by reoxidation of the subunits. F-alpha 2M formed by reaction with a proteinase or ammonium salts was not dissociated under the same conditions, although the interchain disulphide bonds were reduced. If the thiol groups of the quarter-subunits of S-alpha 2M were blocked by carboxymethylation, oxidative reassociation did not occur. Nevertheless treatment of these subunits with methylammonium salts or a proteinase caused the reassembly of half-molecules and intact (F-) tetramers. It is emphasized that F-alpha 2M does not have the properties of a denatured form of the protein... Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:91367

Inactivation of chloroplast H(+)-ATPase by modification of Lys beta 359, Lys alpha 176 and Lys alpha 266.

PubMed

Horbach, M; Meyer, H E; Bickel-Sandkötter, S

1991-09-01

Treatment of isolated, latent chloroplast ATPase with pyridoxal-5-phosphate (pyridoxal-P) in presence of Mg2+ causes inhibition of dithiothreitol-activated plus heat-activated ATP hydrolysis. The amount of [3H]pyridoxal-P bound to chloroplast coupling factor 1 (CF1) was estimated to run up to 6 +/- 1 pyridoxal-P/enzyme, almost equally distributed between the alpha- and beta-subunits. Inactivation, however, is complete after binding of 1.5-2 pyridoxal-P/CF1, suggesting that two covalently modified lysines prevent the activation of the enzyme. ADP as well as ATP in presence of Mg2+ protects the enzyme against inactivation and concomittantly prevents incorporation of a part of the 3H-labeled pyridoxal-P into beta- and alpha-subunits. Phosphate prevents labeling of the alpha-subunit, but has only a minor effect on protection against inactivation. The data indicate a binding site at the interface between the alpha- and beta-subunits. Cleavage of the pyridoxal-P-labeled subunits with cyanogen bromide followed by sequence analysis of the labeled peptides led to the detection of Lys beta 359, Lys alpha 176 and Lys alpha 266, which are closely related to proposed nucleotide-binding regions of the alpha- and beta-subunits.

Characterization and charge distribution of the asparagine-linked oligosaccharides on secreted mouse thyrotropin and free alpha-subunits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gesundheit, N.; Gyves, P.W.; DeCherney, G.S.

1989-06-01

Mouse hemipituitaries in vitro secrete TSH, composed of an alpha-beta heterodimer, as well as excess (free) alpha-subunits. By dual metabolic labeling with (35S)sulfate and (3H)mannose, we have characterized oligosaccharides from secreted TSH alpha, TSH beta, and free alpha-subunits released from the apoprotein by enzymatic deglycosylation. Oligosaccharides from each subunit displayed a distinct anion exchange HPLC profile due to a specific pattern of sialylation and sulfation. Six species were obtained from TSH alpha (with two glycosylation sites), including neutral oligosaccharides as well as those with one or two negative charges. For TSH beta (with one glycosylation site) at least eight oligosaccharidemore » species were noted, representing nearly every permutation of sialylation and sulfation; approximately 30% contained three or more negative charges. Analysis of (3H)mannose-labeled oligosaccharides on Concanavalin-A-agarose showed 85% binding for those from TSH alpha, 70% for free alpha, and 50% for those from TSH beta. These data demonstrate that oligosaccharides from secreted TSH beta were more sialylated and sulfated, consistent with a more complex branching pattern, than those from TSH alpha. Oligosaccharides from free alpha-subunit were more sialylated than those from TSH alpha, and the net negative charge was intermediate between those of TSH alpha and TSH beta. Although great microheterogeneity is present even at the single glycosylation site on the beta-subunit of secreted TSH, a pattern of sialylation and sulfation could be discerned.« less

The alpha2-delta protein: an auxiliary subunit of voltage-dependent calcium channels as a recognized drug target.

PubMed

Thorpe, Andrew J; Offord, James

2010-07-01

Currently, there are two drugs on the market, gabapentin (Neurontin) and pregabalin (Lyrica), that are proposed to exert their therapeutic effect through binding to the alpha2-delta subunit of voltage-sensitive calcium channels. This activity was unexpected, as the alpha2-delta subunit had previously been considered not to be a pharmacological target. In this review, the role of the alpha2-delta subunits is discussed and the mechanism of action of the alpha2-delta ligands in vitro and in vivo is summarized. Finally, new insights into the mechanism of drugs that bind to this protein are discussed.

Identification of a GTP-binding protein. cap alpha. subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.

1988-05-01

Recent molecular cloning of cDNA for the ..cap alpha.. subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein ..cap alpha.. subunit, which they refer to as G/sub z/..cap alpha.., by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ ..cap alpha..-subunit cDNA. The deduced amino acid sequence of G/submore » z/..cap alpha.. is 41-67% identical with those of other known G-protein ..cap alpha.. subunits. However, the 355-residue G/sub z/..cap alpha.. lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein ..cap alpha.. subunits. They suggest that G/sub z/..cap alpha.., which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.« less

X-ray Crystal Structure of Aristolochene Synthase from Aspergillus terreus and Evolution of Templates for the Cyclization of Farnesyl Diphosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shishova,E.; Di Costanzo, L.; Cane, D.

2007-01-01

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 {angstrom} resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the {alpha}-helical class I terpene synthase fold with the active site in the 'open', solvent-exposed conformation. Intriguingly, the 2.15 {angstrom} resolution crystal structure of the complex with Mg{sup 2+}{sub 3}-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the 'closed' conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition frommore » the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved {alpha}-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.« less

Correlation between protein kinase catalytic subunit alpha-1 gene rs13361707 polymorphism and gastric cancer susceptibility in asian populations

PubMed Central

Ni, Jianfeng; Shen, Nan; Tang, Jilei; Ren, Kewei

2017-01-01

A single nucleotide polymorphism (SNP) of the protein kinase catalytic subunit alpha-1 gene (PRKAA1) that confers susceptibility to gastric cancer (GC) was identified by genome-wide association in several case-control studies. However, the results remained controversial and ambiguous. Therefore, we performed a larger meta-analysis to confirm this association. We searched the PubMed, Embase, WanFang, and CNKI databases, without any restriction on language, covering all papers published until Feb 22, 2017. Overall, 14 case-control studies with 14,485 cases and 14,792 controls were retrieved based on the search criteria. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to quantify the strength of the association. Publication bias was assessed by Egger’s and Begg’s tests. We found that the PRKAA1 rs13361707 C/T polymorphism had no association with GC risk in any of the pooled genetic models (for example, the T-allele vs. C-allele allelic contrast model yielded the following estimates: OR = 0.87, 95% CI = 0.73–1.05, Pheterogeneity = 0.000). Furthermore, in analyses stratified by either source of control or geographical origin of subjects, a statistically significant inverse relationship was detected between PRKAA1 rs13361707 C/T polymorphism and GC risk. No obvious evidence of publication bias was detected in the pooled meta-analysis. Furthermore, we observed that individuals carrying T-allele (TT or TC) genotypes had a lower expression of PRKAA1. Our present study indicated that PRKAA1 rs13361707 C/T was not significantly associated with GC risk, despite few positive results in the subgroups. PMID:28978122

Characterization of 3-ketosteroid 9{alpha}-hydroxylase, a Rieske oxygenase in the cholesterol degradation pathway of Mycobacterium tuberculosis.

PubMed

Capyk, Jenna K; D'Angelo, Igor; Strynadka, Natalie C; Eltis, Lindsay D

2009-04-10

KshAB (3-Ketosteroid 9alpha-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O(2). Nevertheless, the reactivity of KshAB with O(2) was low in the presence of 1,4-androstadiene-3,17-dione, with a k(cat)/K(m)(O(2)) of 2450 +/- 80 m(-1) s(-1). The crystallographic structure of KshA, determined to 2.3A(,) revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroid-transforming enzyme of biotechnological and medical importance.

Binding of influenza A virus NS1 protein to the inter-SH2 domain of p85 suggests a novel mechanism for phosphoinositide 3-kinase activation.

PubMed

Hale, Benjamin G; Batty, Ian H; Downes, C Peter; Randall, Richard E

2008-01-18

Influenza A virus NS1 protein stimulates host-cell phosphoinositide 3-kinase (PI3K) signaling by binding to the p85beta regulatory subunit of PI3K. Here, in an attempt to establish a mechanism for this activation, we report further on the functional interaction between NS1 and p85beta. Complex formation was found to be independent of NS1 RNA binding activity and is mediated by the C-terminal effector domain of NS1. Intriguingly, the primary direct binding site for NS1 on p85beta is the inter-SH2 domain, a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. In vitro kinase activity assays, together with protein binding competition studies, reveal that NS1 does not displace p110 from the inter-SH2 domain, and indicate that NS1 can form an active heterotrimeric complex with PI3K. In addition, it was established that residues at the C terminus of the inter-SH2 domain are essential for mediating the interaction between p85beta and NS1. Equivalent residues in p85alpha have previously been implicated in the basal inhibition of p110. However, such p85alpha residues were unable to substitute for those in p85beta with regards NS1 binding. Overall, these data suggest a model by which NS1 activates PI3K catalytic activity by masking a normal regulatory element specific to the p85beta inter-SH2 domain.

Functional and Genomic Analyses of Alpha-Solenoid Proteins

PubMed Central

Fournier, David; Palidwor, Gareth A.; Shcherbinin, Sergey; Szengel, Angelika; Schaefer, Martin H.; Perez-Iratxeta, Carol; Andrade-Navarro, Miguel A.

2013-01-01

Alpha-solenoids are flexible protein structural domains formed by ensembles of alpha-helical repeats (Armadillo and HEAT repeats among others). While homology can be used to detect many of these repeats, some alpha-solenoids have very little sequence homology to proteins of known structure and we expect that many remain undetected. We previously developed a method for detection of alpha-helical repeats based on a neural network trained on a dataset of protein structures. Here we improved the detection algorithm and updated the training dataset using recently solved structures of alpha-solenoids. Unexpectedly, we identified occurrences of alpha-solenoids in solved protein structures that escaped attention, for example within the core of the catalytic subunit of PI3KC. Our results expand the current set of known alpha-solenoids. Application of our tool to the protein universe allowed us to detect their significant enrichment in proteins interacting with many proteins, confirming that alpha-solenoids are generally involved in protein-protein interactions. We then studied the taxonomic distribution of alpha-solenoids to discuss an evolutionary scenario for the emergence of this type of domain, speculating that alpha-solenoids have emerged in multiple taxa in independent events by convergent evolution. We observe a higher rate of alpha-solenoids in eukaryotic genomes and in some prokaryotic families, such as Cyanobacteria and Planctomycetes, which could be associated to increased cellular complexity. The method is available at http://cbdm.mdc-berlin.de/~ard2/. PMID:24278209

Cross-linking of hCG to luteal receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, T.H.; Ji, I.

1985-01-01

Photoaffinity labeling of the lutropin/choriogonadotropin (LH/hCG) receptor system on porcine granulosa cells has demonstrated that both the ..cap alpha.. and ..beta.. subunits of hCG directly photoaffinity label the hormone receptor. Three new bands appear on SDS-PAGE as a consequence of photoaffinity labeling by each subunit: the molecular weights of the three bands (106K, 88K, and 83K) produced by the subunit are larger by approximately 10K than those of the three bands (96K, 76K, and 73K) labeled by the ..cap alpha.. subunit. Although it could be a coincidence that the molecular weight of the ..beta.. subunit is approximately 10K larger thanmore » that of the ..cap alpha.. subunit, the similarity in these differences suggests the possibility that both the ..cap alpha.. and ..beta.. subunits have labeled the same polypeptides.« less

Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1).

PubMed

Tabish, M; Clegg, R A; Rees, H H; Fisher, M J

1999-04-01

The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.

Cloning and sequence analysis of a cDNA encoding the alpha-subunit of mouse beta-N-acetylhexosaminidase and comparison with the human enzyme.

PubMed Central

Beccari, T; Hoade, J; Orlacchio, A; Stirling, J L

1992-01-01

cDNAs encoding the mouse beta-N-acetylhexosaminidase alpha-subunit were isolated from a mouse testis library. The longest of these (1.7 kb) was sequenced and showed 83% similarity with the human alpha-subunit cDNA sequence. The 5' end of the coding sequence was obtained from a genomic DNA clone. Alignment of the human and mouse sequences showed that all three putative N-glycosylation sites are conserved, but that the mouse alpha-subunit has an additional site towards the C-terminus. All eight cysteines in the human sequence are conserved in the mouse. There are an additional two cysteines in the mouse alpha-subunit signal peptide. All amino acids affected in Tay-Sachs-disease mutations are conserved in the mouse. Images Fig. 1. PMID:1379046

Spontaneous quaternary and tertiary T-R transitions of human hemoglobin in molecular dynamics simulation.

PubMed

Hub, Jochen S; Kubitzki, Marcus B; de Groot, Bert L

2010-05-06

We present molecular dynamics simulations of unliganded human hemoglobin (Hb) A under physiological conditions, starting from the R, R2, and T state. The simulations were carried out with protonated and deprotonated HC3 histidines His(beta)146, and they sum up to a total length of 5.6 micros. We observe spontaneous and reproducible T-->R quaternary transitions of the Hb tetramer and tertiary transitions of the alpha and beta subunits, as detected from principal component projections, from an RMSD measure, and from rigid body rotation analysis. The simulations reveal a marked asymmetry between the alpha and beta subunits. Using the mutual information as correlation measure, we find that the beta subunits are substantially more strongly linked to the quaternary transition than the alpha subunits. In addition, the tertiary populations of the alpha and beta subunits differ substantially, with the beta subunits showing a tendency towards R, and the alpha subunits showing a tendency towards T. Based on the simulation results, we present a transition pathway for coupled quaternary and tertiary transitions between the R and T conformations of Hb.

Alpha subunit of glycoprotein hormones in the sera of acromegalic patients and its mRNA in the tumors.

PubMed

Machiavelli, G A; Artese, R; Benencia, H; Bruno, O; Guerra, L; Basso, A; Burdman, J A

1999-04-01

Within a population of 16 pituitary adenomas we found high levels of glycoprotein alpha subunits in the sera of patients with somatotrophic tumors. This finding was correlated with the presence of mRNA alpha subunit in these tumors indicating the adenomas themselves as the origin of the circulating alpha-subunit. Synthesis of these two hormones, which are chemically very different, by the same tumor cells indicates a high degree of differentiation of these cells. We are unable at this time to conclusively correlate differentiation of these tumors aggressively.

Mechanism of repression of the inhibin alpha-subunit gene by inducible 3',5'-cyclic adenosine monophosphate early repressor.

PubMed

Burkart, Anna D; Mukherjee, Abir; Mayo, Kelly E

2006-03-01

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.

Inherent conformational flexibility of F1-ATPase α-subunit.

PubMed

Hahn-Herrera, Otto; Salcedo, Guillermo; Barril, Xavier; García-Hernández, Enrique

2016-09-01

The core of F1-ATPase consists of three catalytic (β) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in β-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3β3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and β-subunits show on Mg(II) for recognizing ATP. Copyright © 2016 Elsevier B.V. All rights reserved.

Coexpression of alpha and beta subunits of the rod cyclic GMP-gated channel restores native sensitivity to cyclic AMP: role of D604/N1201.

PubMed Central

Pagès, F; Ildefonse, M; Ragno, M; Crouzy, S; Bennett, N

2000-01-01

Coexpression of the betawt and alphawt subunits of the bovine rod channel restores two characteristics of the native channels: higher sensitivity to cAMP and potentiation of cGMP-induced currents by low cAMP concentrations. To test whether the increased sensitivity to cAMP is due to the uncharged nature of the asparagine residue (N1201) situated in place of aspartate D604 in the beta subunit as previously suggested (, Neuron. 15:619-625), we compared currents from wild-type (alphawt and alphawt/betawt) and from mutated channels (alphaD604N, alphaD604N/betawt, and alphawt/betaN1201D). The results show that the sensitivity to cAMP and cAMP potentiation is partly but not entirely determined by the charge of residue 1201 in the beta subunit. The D604N mutation in the alpha subunit and, to a lesser extent, coexpression of the betawt subunit with the alphawt subunit reduce the open probability for cGMP compared to that of the alphawt channel. Interpretation of the data with the MWC allosteric model (model of Monod, Wyman, Changeux;, J. Mol. Biol. 12:88-118) suggests that the D604N mutation in the alpha subunits and coassembly of alpha and beta subunits alter the free energy of gating by cAMP more than that of cAMP binding. PMID:10692312

Rates of processing of the high mannose oligosaccharide units at the three glycosylation sites of mouse thyrotropin and the two sites of free alpha-subunits.

PubMed

Miura, Y; Perkel, V S; Magner, J A

1988-09-01

We have determined the structures of high mannose (Man) oligosaccharide units at individual glycosylation sites of mouse TSH. Mouse thyrotropic tumor tissue was incubated with D-[2-3H]Man with or without [14C]tyrosine ([14C] Tyr) for 2, 3, or 6 h, and for a 3-h pulse followed by a 2-h chase. TSH heterodimers or free alpha-subunits were obtained from homogenates using specific antisera. After reduction and alkylation, subunits were treated with trypsin. The tryptic fragments were then loaded on a reverse phase HPLC column to separate tryptic fragments bearing labeled oligosaccharides. The N-linked oligosaccharides were released with endoglycosidase-H and analyzed by paper chromatography. Man9GlcNac2 and Man8GlcNac2 units predominated at each time point and at each specific glycosylation site, but the processing of high Man oligosaccharides differed at each glycosylation site. The processing at Asn23 of TSH beta-subunits was slower than that at Asn56 or Asn82 of alpha-subunits. The processing at Asn82 was slightly faster than that at Asn56 for both alpha-subunits of TSH heterodimers and free alpha-subunits. The present study demonstrates that the early processing of oligosaccharides differs at the individual glycosylation sites of TSH and free alpha-subunits, perhaps because of local conformational differences.

Structure of a catalytic dimer of the α- and β-subunits of the F-ATPase from Paracoccus denitrificans at 2.3 Å resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morales-Ríos, Edgar; Montgomery, Martin G.; Leslie, Andrew G. W.

2015-09-23

The structure of the αβ heterodimer of the F-ATPase from the α-proteobacterium P. denitrificans has been determined at 2.3 Å resolution. It corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases. The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas manymore » others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F{sub 1} domain of the enzyme. This domain is a complex of three α-subunits and three β-subunits, and one copy of each of the γ-, δ- and ∊-subunits. Attempts to crystallize the F{sub 1}–ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and β-subunits only. Its structure was determined to 2.3 Å resolution and consists of a heterodimer of one α-subunit and one β-subunit. It has no bound nucleotides, and it corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized.« less

Cloning, Characterization, and Expression Analysis of the Novel Acetyltransferase Retrogene Ard1b in the Mouse1

PubMed Central

Pang, Alan Lap-Yin; Peacock, Stephanie; Johnson, Warren; Bear, Deborah H.; Rennert, Owen M.; Chan, Wai-Yee

2009-01-01

N-alpha-terminal acetylation is a modification process that occurs cotranslationally on most eukaryotic proteins. The major enzyme responsible for this process, N-alpha-terminal acetyltransferase, is composed of the catalytic subunit ARD1A and the auxiliary subunit NAT1. We cloned, characterized, and studied the expression pattern of Ard1b (also known as Ard2), a novel homolog of the mouse Ard1a. Comparison of the genomic structures suggests that the autosomal Ard1b is a retroposed copy of the X-linked Ard1a. Expression analyses demonstrated a testis predominance of Ard1b. A reciprocal expression pattern between Ard1a and Ard1b is also observed during spermatogenesis, suggesting that Ard1b is expressed to compensate for the loss of Ard1a starting from meiosis. Both ARD1A and ARD1B can interact with NAT1 to constitute a functional N-alpha-terminal acetyltransferase in vitro. The expression of ARD1B protein can be detected in mouse testes but is delayed until the first appearance of round spermatids. In a cell culture model, the inclusion of the long 3′ untranslated region of Ard1b leads to reduction of luciferase reporter activity, which implicates its role in translational repression of Ard1b during spermatogenesis. Our results suggest that ARD1B may have an important role in the later course of the spermatogenic process. PMID:19246321

Cloning, characterization, and expression analysis of the novel acetyltransferase retrogene Ard1b in the mouse.

PubMed

Pang, Alan Lap-Yin; Peacock, Stephanie; Johnson, Warren; Bear, Deborah H; Rennert, Owen M; Chan, Wai-Yee

2009-08-01

N-alpha-terminal acetylation is a modification process that occurs cotranslationally on most eukaryotic proteins. The major enzyme responsible for this process, N-alpha-terminal acetyltransferase, is composed of the catalytic subunit ARD1A and the auxiliary subunit NAT1. We cloned, characterized, and studied the expression pattern of Ard1b (also known as Ard2), a novel homolog of the mouse Ard1a. Comparison of the genomic structures suggests that the autosomal Ard1b is a retroposed copy of the X-linked Ard1a. Expression analyses demonstrated a testis predominance of Ard1b. A reciprocal expression pattern between Ard1a and Ard1b is also observed during spermatogenesis, suggesting that Ard1b is expressed to compensate for the loss of Ard1a starting from meiosis. Both ARD1A and ARD1B can interact with NAT1 to constitute a functional N-alpha-terminal acetyltransferase in vitro. The expression of ARD1B protein can be detected in mouse testes but is delayed until the first appearance of round spermatids. In a cell culture model, the inclusion of the long 3' untranslated region of Ard1b leads to reduction of luciferase reporter activity, which implicates its role in translational repression of Ard1b during spermatogenesis. Our results suggest that ARD1B may have an important role in the later course of the spermatogenic process.

GTP analogues promote release of the alpha subunit of the guanine nucleotide binding protein, Gi2, from membranes of rat glioma C6 BU1 cells.

PubMed Central

Milligan, G; Mullaney, I; Unson, C G; Marshall, L; Spiegel, A M; McArdle, H

1988-01-01

The major pertussis-toxin-sensitive guanine nucleotide-binding protein of rat glioma C6 BU1 cells corresponded immunologically to Gi2. Antibodies which recognize the alpha subunit of this protein indicated that it has an apparent molecular mass of 40 kDa and a pI of 5.7. Incubation of membranes of these cells with guanosine 5'-[beta gamma-imido]triphosphate, or other analogues of GTP, caused release of this polypeptide from the membrane in a time-dependent manner. Analogues of GDP or of ATP did not mimic this effect. The GTP analogues similarly caused release of the alpha subunit of Gi2 from membranes of C6 cells in which this G-protein had been inactivated by pretreatment with pertussis toxin. The beta subunit was not released from the membrane under any of these conditions, indicating that the release process was a specific response to the dissociation of the G-protein after binding of the GTP analogue. Similar nucleotide profiles for release of the alpha subunits of forms of Gi were noted for membranes of both the neuroblastoma x glioma hybrid cell line NG108-15 and of human platelets. These data provide evidence that: (1) pertussis-toxin-sensitive G-proteins, in native membranes, do indeed dissociate into alpha and beta gamma subunits upon activation; (2) the alpha subunit of 'Gi-like' proteins need not always remain in intimate association with the plasma membrane; and (3) the alpha subunit of Gi2 can still dissociate from the beta/gamma subunits after pertussis-toxin-catalysed ADP-ribosylation. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:3140801

Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1alpha-mediated localization in living cells.

PubMed

Martin, Brent R; Deerinck, Thomas J; Ellisman, Mark H; Taylor, Susan S; Tsien, Roger Y

2007-09-01

The tetracysteine sequence YRECCPGCCMWR fused to the N terminus of green fluorescent protein (GFP) self-aggregates upon biarsenical labeling in living cells or in vitro. Such dye-triggered aggregates form temperature-dependent morphologies and are dispersed by photobleaching. Fusion of the biarsenical aggregating GFP to the regulatory (R) or catalytic (C) subunit of PKA traps intact holoenzyme in compact fluorescent puncta upon biarsenical labeling. Contrary to the classical model of PKA activation, elevated cAMP does not allow RIalpha and Calpha to diffuse far apart unless the pseudosubstrate inhibitor PKI or locally concentrated substrate is coexpressed. However, RIIalpha releases Calpha upon elevated cAMP alone, dependent on autophosphorylation of the RIIalpha inhibitory domain. DAKAP1alpha overexpression induced R and C outer mitochondrial colocalization and showed similar regulation. Overall, effective separation of type I PKA is substrate dependent, whereas type II PKA dissociation relies on autophosphorylation.

Integrin distributions in renal cell carcinomas of various grades of malignancy.

PubMed Central

Korhonen, M.; Laitinen, L.; Ylänne, J.; Koukoulis, G. K.; Quaranta, V.; Juusela, H.; Gould, V. E.; Virtanen, I.

1992-01-01

We studied 41 renal cell carcinomas, classified according to histologic grades G1 through G3, by indirect immunofluorescence microscopy using a panel of monoclonal antibodies (MAb) against various integrin subunits, and the basement membrane (BM) components laminin and collagen type IV. Selected cases also were immunostained using the avidin-biotin-complex method. The alpha 3 and beta 1 integrin subunits were detected in tumor cells of all the carcinomas. All G1 carcinomas, like normal tubular epithelial cells, expressed the alpha 6 subunit, whereas it was lacking in 20% and 40% of G2 and G3 carcinomas, respectively. Furthermore, when alpha 6 was expressed, a lack of basally polarized organization of the subunit, coupled with disorganization of the BM components, correlated with histologic grade. Another feature that appeared to characterize the more anaplastic tumors was their high level (80%) of the alpha v subunit expression as compared with its absence in the G1 carcinomas. Stromal myofibroblasts, identified by double-labeling with anti-myosin, were often characterized by the expression of the alpha 1, alpha 3, alpha 5 and beta 1 subunits. These results indicate that changes in integrin expression in renal cell carcinomas may be correlated with their degree of histologic malignancy. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1443050

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

PubMed

Avni, A; Avital, S; Gromet-Elhanan, Z

1991-04-25

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.

Amino acid sequence of the human fibronectin receptor

PubMed Central

1987-01-01

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+- binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors. PMID:2958481

Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1.

PubMed

Kida, Hiroshi; Sugano, Yuri; Iizuka, Ryo; Fujihashi, Masahiro; Yohda, Masafumi; Miki, Kunio

2008-11-14

Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of alpha and beta subunits and forms a "jellyfish-like" structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two alpha and two beta subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of alpha (alpha1 and alpha2) and beta subunits (beta1 and beta2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD beta1 subunit at 1.9 A resolution and its functional analysis. TsPFD beta1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The beta hairpin linkers of beta1 subunits assemble to form a beta barrel "body" around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the beta1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric beta1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD beta1 subunits act as molecular chaperones in living cells of some archaea.

Direct catalytic asymmetric alpha-amination of aldehydes.

PubMed

List, Benjamin

2002-05-22

The first direct catalytic asymmetric alpha-amination of aldehydes is described herein. alpha-Unbranched aldehydes react in this novel proline-catalyzed reaction with dialkyl azodicarboxylates to give alpha-amino aldehydes in excellent yields and enantioselectivities.

Structure of N-acetyl-[beta]-D-glucosaminidase (GcnA) from the Endocarditis Pathogen Streptococcus gordonii and its Complex with the Mechanism-based Inhibitor NAG-thiazoline

DOE Office of Scientific and Technical Information (OSTI.GOV)

Langley, David B.; Harty, Derek W.S.; Jacques, Nicholas A.

2008-09-17

The crystal structure of GcnA, an N-acetyl-{beta}-D-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal {alpha}-helical domain has not been observed previously and forms a large dimerization interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical ({beta}/{alpha}){sub 8} TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a familymore » 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-{beta}-D-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.« less

Genetic ablation of the alpha 6-integrin subunit in Tie1Cre mice enhances tumour angiogenesis.

PubMed

Germain, Mitchel; De Arcangelis, Adèle; Robinson, Stephen D; Baker, Marianne; Tavora, Bernardo; D'Amico, Gabriela; Silva, Rita; Kostourou, Vassiliki; Reynolds, Louise E; Watson, Alan; Jones, J Louise; Georges-Labouesse, Elisabeth; Hodivala-Dilke, Kairbaan

2010-02-01

Laminins are expressed highly in blood vessel basement membranes and have been implicated in angiogenesis. alpha6beta1- and alpha6beta4-integrins are major receptors for laminins in endothelial cells, but the precise role of endothelial alpha6-integrin in tumour angiogenesis is not clear. We show that blood vessels in human invasive ductal carcinoma of the breast have decreased expression of the alpha6-integrin-subunit when compared with normal breast tissue. These data suggest that a decrease in alpha6-integrin-subunit expression in endothelial cells is associated with tumour angiogenesis. To test whether the loss of the endothelial alpha6-integrin subunit affects tumour growth and angiogenesis, we generated alpha6fl/fl-Tie1Cre+ mice and showed that endothelial deletion of alpha6-integrin is sufficient to enhance tumour size and tumour angiogenesis in both murine B16F0 melanoma and Lewis cell lung carcinoma. Mechanistically, endothelial alpha6-integrin deficiency elevated significantly VEGF-mediated angiogenesis both in vivo and ex vivo. In particular, alpha6-integrin-deficient endothelial cells displayed increased levels of VEGF-receptor 2 (VEGFR2) and VEGF-mediated downstream ERK1/2 activation. By developing the first endothelial-specific alpha6-knockout mice, we show that the expression of the alpha6-integrin subunit in endothelial cells acts as a negative regulator of angiogenesis both in vivo and ex vivo. Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits.

PubMed

Snyder, P M; Cheng, C; Prince, L S; Rogers, J C; Welsh, M J

1998-01-09

Members of the DEG/ENaC protein family form ion channels with diverse functions. DEG/ENaC subunits associate as hetero- and homomultimers to generate channels; however the stoichiometry of these complexes is unknown. To determine the subunit stoichiometry of the human epithelial Na+ channel (hENaC), we expressed the three wild-type hENaC subunits (alpha, beta, and gamma) with subunits containing mutations that alter channel inhibition by methanethiosulfonates. The data indicate that hENaC contains three alpha, three beta, and three gamma subunits. Sucrose gradient sedimentation of alphahENaC translated in vitro, as well as alpha-, beta-, and gammahENaC coexpressed in cells, was consistent with complexes containing nine subunits. FaNaCh and BNC1, two related DEG/ENaC channels, produced complexes of similar mass. Our results suggest a novel nine-subunit stoichiometry for the DEG/ENaC family of ion channels.

Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Meisheng; Tran, V.T.; Fong, H.K.W.

1991-05-01

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha}more » protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.« less

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1.

PubMed

Iizuka, Ryo; Sugano, Yuri; Ide, Naoki; Ohtaki, Akashi; Yoshida, Takao; Fujiwara, Shinsuke; Imanaka, Tadayuki; Yohda, Masafumi

2008-03-28

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two alpha subunits and four beta subunits with the structure of a double beta-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (alpha1, alpha2, beta1, and beta2) from T. KS-1. All of them (alpha1-beta1, alpha2-beta1, alpha1-beta2, and alpha2-beta2) exist as alpha(2)beta(4) heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the beta1 subunit interacted with the chaperonins more strongly than those with the beta2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.

Recombination and mutation of class II histocompatibility genes in wild mice.

PubMed

Wakeland, E K; Darby, B R

1983-12-01

We have compared the tryptic peptide fingerprints of the A alpha, A beta, E alpha, and E beta subunits encoded by four wild-derived H-2 complexes expressing A molecules closely related to Ak. The A molecules encoded by these Ak-related mice have A alpha and A beta subunits that differ from A alpha k and A beta k by less than 10% of their tryptic peptides. Comparisons among the four wild-derived A molecules suggested that these contemporary A alpha and A beta alleles arose by sequential mutational events from common ancestor A alpha and A beta alleles. These results suggest that A alpha and A beta may co-evolve as an A beta A alpha gene duplex in wild mice. Tryptic peptide fingerprint comparisons of the E beta gene linked to these Ak-related A beta A alpha gene duplexes indicate that two encode E beta d-like subunits, whereas another encodes an E beta s-like subunit. These results strongly suggest that the A beta A alpha duplex and E beta recombine in wild mouse populations. The significantly different evolutionary patterns exhibited by the class II genes encoding A vs E molecules are discussed.

Localization of nuclear subunits of cyclic AMP-dependent protein kinase by the immunocolloidal gold method

PubMed Central

1985-01-01

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP- dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density. PMID:2993318

Quantitative RT-PCR for inhibin/activin subunits: measurements of rat hypothalamic and ovarian inhibin/activin subunit mRNAs during the estrous cycle.

PubMed

Murata, T; Takizawa, T; Funaba, M; Fujimura, H; Murata, E; Takahashi, M; Torii, K

1997-02-01

Inhibins (alpha-beta(A) and alpha-beta(B)) and activins (beta(A)-beta(A), beta(A)-beta(B) and beta(B)-beta(B)) were originally isolated from ovarian follicular fluids as FSH secretion modifiers. Inhibin/activin subunits, alpha, beta(A) and beta(B), are widely distributed in several tissues, including gonads and brain, and inhibins and activins have been reported to be involved in ovarian or hypothalamic functions. In this study, we established and employed a competitive RT-PCR assay system for rat inhibin/activin subunits by capillary electrophoresis to determine rat hypothalamic and ovarian inhibin/activin subunit mRNA levels during the estrous cycle. Linearity of standards for alpha, beta(A), and beta(B) subunit assays were between 0.01-0.3 amol, 0.003-0.09 amol and 0.002-0.02 amol of each fragment DNA as a standard, respectively. Hypothalamic beta(A) subunit mRNA during the estrous morning (1000 h) tended to be increased compared with that of the proestrous evening (1700 h), although they were not significantly different. Ovarian alpha subunit mRNA levels tended to be increased during the proestrous morning (1000 h) and were significantly increased in the proestrous evening (1700 h), compared with diestrus and estrus (P < 0.05). Ovarian beta(A) subunit mRNA was also significantly higher in the proestrous evening, compared with diestrus and estrus (P < 0.05), but in the case of beta(B) subunit mRNA there was no difference among diestrus, proestrus and estrus. We thus established a sensitive competitive RT-PCR system for the measurement of inhibin/activin alpha, beta(A) and beta(B) subunits, and this assay system would be helpful for the study of inhibin/activin action in brain and other tissues where these factors are expressed at low levels.

SnRK1 is differentially regulated in the cotyledon and embryo axe of bean (Phaseolus vulgaris L) seeds.

PubMed

Coello, Patricia; Martínez-Barajas, Eleazar

2014-07-01

SnRK1 activity is developmentally regulated in bean seeds and exhibits a transient increase with the highest value at 20 days after anthesis (DAA), which coincides with the beginning of protein and starch accumulation. The catalytic subunit of SnRK1 shows a consistent decrease throughout the seed development period. However, by 15 DAA a significant proportion of the catalytic subunit appears phosphorylated. The increase in activity and phosphorylation of the catalytic subunit coincides with a decrease in hexoses. However, SnRK1 activity is differentially regulated in the cotyledon and embryo axe, where a larger proportion of the catalytic subunit is phosphorylated. SnRK1 obtained from endosperm extract is inhibited by T6P and to a lesser extent by ADPG and UDPG, whereas the enzyme isolated from embryo is virtually insensitive to T6P but exhibits some inhibition by ADPG and UDPG. In cotyledon extracts, the effects of T6P and ADPG on SnRK1 activity are additive, whereas in embryo extract, T6P inhibits the enzyme only when ADPG is present. After fractionation on Sephacryl-S300, SnRK1 activity obtained from cotyledon extracts is detected as a single peak associated with a molecular weight of 250 kDa whereas that obtained form embryo axe extracts detected as 2 peaks associated with molecular weight of 250 and 180 kDa. In both cases, the catalytic subunit exhibits a wide distribution but is concentrated in the fractions with the highest activity. To analyse the composition of the complex, cotyledon and embryo extracts were treated with a reversible crosslinker (DSP). DSP induced the formation of complexes with molecular weights of 97 and 180 kDa in the cotyledon and embryo extracts, respectively. Since all the phosphorylated catalytic subunit is present in the complexes induced by DSP, it appears that the phosphorylation favors its interaction with other proteins. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Unexpected high digestion rate of cooked starch by the Ct-Maltase-Glucoamylase small intestine mucosal alpha-glucosidase subunit

USDA-ARS?s Scientific Manuscript database

For starch digestion to glucose, two luminal alpha-amylases and four gut mucosal alpha-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal alpha-glucosidases on cooked (gelatinized) starch. Gelatinized ...

Characterization of inhibin forms and their measurement by an inhibin alpha-subunit ELISA in serum from postmenopausal women with ovarian cancer.

PubMed

Robertson, D M; Stephenson, T; Pruysers, E; McCloud, P; Tsigos, A; Groome, N; Mamers, P; Burger, H G

2002-02-01

The aim of this study was to characterize the molecular wt forms of inhibins A and B and its free alpha-subunit present in serum from women with ovarian cancer as a basis for developing improved monoclonal antibody-based inhibin assays for monitoring ovarian cancer. Three new inhibin alpha-subunit (alphaC) ELISAs were developed using monoclonal antibodies directed to three nonoverlapping peptide regions of the alphaC region of the inhibin alpha-subunit. To characterize serum inhibin molecular wt forms present in women with ovarian cancer, existing inhibin immunoassays (inhibin A, inhibin B, and pro-alphaC) and the new alphaC ELISAs were applied to sera from women with granulosa cell tumors and mucinous carcinomas previously fractionated using a combined immunoaffinity chromatography, preparative SDS-PAGE, and electroelution procedure. The distribution and molecular size of dimeric inhibins and alpha-subunit detected were consistent with known mol wt forms of inhibins A and B and inhibin alpha-subunit and their precursor forms present in serum and follicular fluid from healthy women. The alphaC ELISAs recognized all known forms of inhibin and the free inhibin alpha-subunit, although differences between alphaC ELISAs were observed in their ability to detect high mol wt forms. To assess which of the alphaC ELISAs was preferred in application to ovarian cancer, the alphaC ELISAs were applied to serum from a range of normal postmenopausal women (n = 61) and postmenopausal women (n = 152) with ovarian (serous, mucinous, endometrioid, clear cell carcinomas, and granulosa cell tumors) and nonovarian (breast and colon) cancers. Despite differences in their ability to detect high mol wt forms of inhibin, the alphaC ELISAs showed similar sensitivity (i.e. proportion of cancer patients correctly detected) and specificity (proportion of controls correctly detected) indexes in the detection of mucinous carcinomas (84% and 95%) and granulosa cell tumors (100% and 95%) compared with earlier inhibin RIA or polyclonal antibody-based immunofluorometric assays. A combination of the alphaC ELISAs with the CA125 assay, an ovarian tumor marker that has a high sensitivity and specificity for other ovarian cancers (serous, clear cell, and endometrioid), resulted in an increase in sensitivity/specificity indexes (95% and 95%) for the all ovarian cancer group. These new monoclonal antibody-based inhibin alphaC ELISAs now provide practical and sensitive assays suitable for evaluation as diagnostic tests for monitoring ovarian cancers.

Rotation of Subunits During Catalysis by Escherichia coli F_1-ATPase

NASA Astrophysics Data System (ADS)

Duncan, Thomas M.; Bulygin, Vladimir V.; Zhou, Yuantai; Hutcheon, Marcus L.; Cross, Richard L.

1995-11-01

During oxidative and photo-phosphorylation, F_0F_1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F_0F_1. Guided by a recent, high-resolution structure for bovine F_1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central γ subunit relative to the three catalytic β subunits in soluble F_1 from Escherichia coli. In the bovine F_1 structure, a specific point of contact between the γ subunit and one of the three catalytic β subunits includes positioning of the homolog of E. coli γ-subunit C87 (γC87) close to the β-subunit 380DELSEED386 sequence. A βD380C mutation allowed us to induce formation of a specific disulfide bond between β and γC87 in soluble E. coli F_1. Formation of the crosslink inactivated βD380C-F_1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked βD380C-F_1, we incorporated radiolabeled β subunits into the two noncrosslinked β-subunit positions of F_1. After reduction of the initial nonradio-active β-γ crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled β subunits with γC87 upon reoxidation. The results demonstrate that γ subunit rotates relative to the β subunits during catalysis.

Kinetics of acrylodan-labelled cAMP-dependent protein kinase catalytic subunit denaturation.

PubMed

Kivi, Rait; Loog, Mart; Jemth, Per; Järv, Jaak

2013-10-01

Fluorescence spectroscopy was used to study denaturation of cAMP-dependent protein kinase catalytic subunit labeled with an acrylodan moiety. The dye was covalently bound to a cystein residue introduced into the enzyme by replacement of arginine in position 326 in the native sequence, located near the enzyme active center. This labeling had no effect on catalytic activity of the enzyme, but provided possibility to monitor changes in protein structure through measuring the fluorescence spectrum of the dye, which is sensitive to changes in its environment. This method was used to monitor denaturation of the protein kinase catalytic subunit and study the kinetics of this process as well as influence of specific ligands on stability of the protein. Stabilization of the enzyme structure was observed in the presence of adenosine triphosphate, peptide substrate RRYSV and inhibitor peptide PKI[5-24].

The characteristics of the (alpha V371C)3(beta R337C)3 gamma double mutant subcomplex of the TF1-ATPase indicate that the catalytic site at the alpha TP-beta TP interface with bound MgADP in crystal structures of MF1 represents a catalytic site containing inhibitory MgADP.

PubMed

Bandyopadhyay, Sanjay; Muneyuki, Eiro; Allison, William S

2005-02-22

In the MF(1) crystal structure with the MgADP-fluoroaluminate complex bound to two catalytic sites [Menz, R. I., Walker, J. E., and Leslie, A. G. W. (2001) Cell 106, 331-341], the guanidinium of betaR(337) is within 2.9 A of the alpha-oxygen of alphaS(370) and 3.7 A of a methyl group of alphaV(371) at the alpha(E)-beta(HC) interface. To examine the functional role of this contact, the (alphaV(371)C)(3)(betaR(337)C)(3)gamma subcomplex of the TF(1)-ATPase was prepared and characterized. Steady state ATPase activity of the reduced double-mutant is 30% of that of the wild type. Inactivation of the double mutant containing empty catalytic sites or MgADP bound to one catalytic site with CuCl(2) cross-linked two alpha-beta pairs, whereas a single alpha-beta pair cross-linked when at least two catalytic sites contained MgADP. The reduced double mutant hydrolyzed substoichiometric ATP 100-fold more rapidly than the enzyme containing two cross-linked alpha-beta pairs. Addition of AlCl(3) and NaF to the reduced double mutant after incubation with stoichiometric MgADP or 200 microM MgADP irreversibly inactivated the steady state ATPase activity with rate constants of 1.5 x10(-2) and 4.1 x 10(-2) min(-1), respectively. In contrast, addition of AlCl(3) and NaF to the cross-linked enzyme after incubation with stoichiometric or 200 microM MgADP irreversibly inactivated ATPase activity with a common rate constant of approximately 10(-4) min(-1). Correlation of these results with crystal structures of MF(1) suggests that the catalytic site at the alpha(TP)-beta(TP) interface is loaded first upon addition of nucleotides to nucleotide-depleted F(1)-ATPases and that the catalytic site at the alpha(TP)-beta(TP) interface with bound MgADP in crystal structures represents a catalytic site containing inhibitory MgADP.

beta subunits influence the biophysical and pharmacological differences between P- and Q-type calcium currents expressed in a mammalian cell line.

PubMed

Moreno, H; Rudy, B; Llinás, R

1997-12-09

Human epithelial kidney cells (HEK) were prepared to coexpress alpha1A, alpha2delta with different beta calcium channel subunits and green fluorescence protein. To compare the calcium currents observed in these cells with the native neuronal currents, electrophysiological and pharmacological tools were used conjointly. Whole-cell current recordings of human epithelial kidney alpha1A-transfected cells showed small inactivating currents in 80 mM Ba2+ that were relatively insensitive to calcium blockers. Coexpression of alpha1A, betaIb, and alpha2delta produced a robust inactivating current detected in 10 mM Ba2+, reversibly blockable with low concentration of omega-agatoxin IVA (omega-Aga IVA) or synthetic funnel-web spider toxin (sFTX). Barium currents were also supported by alpha1A, beta2a, alpha2delta subunits, which demonstrated the slowest inactivation and were relatively insensitive to omega-Aga IVA and sFTX. Coexpression of beta3 with the same combination as above produced inactivating currents also insensitive to low concentration of omega-Aga IVA and sFTX. These data indicate that the combination alpha1A, betaIb, alpha2delta best resembles P-type channels given the rate of inactivation and the high sensitivity to omega-Aga IVA and sFTX. More importantly, the specificity of the channel blocker is highly influenced by the beta subunit associated with the alpha1A subunit.

cap alpha. /sub i/-3 cDNA encodes the. cap alpha. subunit of G/sub k/, the stimulatory G protein of receptor-regulated K/sup +/ channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Codina, J.; Olate, J.; Abramowitz, J.

1988-05-15

cDNA cloning has identified the presence in the human genome of three genes encoding ..cap alpha.. subunits of pertussis toxin substrates, generically called G/sub i/. They are named ..cap alpha../sub i/-1, ..cap alpha../sub i/-2 and ..cap alpha../sub i/-3. However, none of these genes has been functionally identified with any of the ..cap alpha.. subunits of several possible G proteins, including pertussis toxin-sensitive G/sub p/'s, stimulatory to phospholipase C or A/sub 2/, G/sub i/, inhibitory to adenylyl cyclase, or G/sub k/, stimulatory to a type of K/sup +/ channels. The authors now report the nucleotide sequence and the complete predicted aminomore » acid sequence of human liver ..cap alpha../sub i/-3 and the partial amino acid sequence of proteolytic fragments of the ..cap alpha.. subunit of human erythrocyte G/sub k/. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of ..cap alpha../sub i/-3, thus identifying it as ..cap alpha../sub k/. The probable identity of ..cap alpha../sub i/-1 with ..cap alpha../sub p/ and possible roles for ..cap alpha../sub i/-2, as well as additional roles for ..cap alpha../sub i/-1 and ..cap alpha../sub i/-3 (..cap alpha../sub k/) are discussed.« less

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ε

PubMed Central

Zahurancik, Walter J.; Baranovskiy, Andrey G.; Tahirov, Tahir H.; Suo, Zucai

2015-01-01

Numerous genetic studies have provided compelling evidence to establish DNA polymerase ε (Polε) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polε is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′ → 5′ exonuclease domain common to many replicative polymerases. In addition, Polε possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polε heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polε in vitro. However, similar studies of the human Polε heterote-tramer (hPolε) have been limited by the difficulty of obtaining hPolε in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolε from insect host cells has allowed for isolation of greater amounts of active hPolε, thus enabling a more detailed kinetic comparison between hPolε and an active N-terminal fragment of the hPolε catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolε. We observe that the small subunits increase DNA binding by hPolε relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolε is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolε and sway hPolε toward DNA synthesis rather than proofreading. PMID:25684708

GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

NASA Technical Reports Server (NTRS)

Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

1995-01-01

The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of schizophrenics, the previously reported upregulation of muscimol binding sites and downregulation of benzodiazepine binding sites in the prefrontal and adjacent cingulate cortex of schizophrenics are possibly due to posttranscriptional modifications of mRNAs and their translated polypeptides.

Functional analysis of Gossypium hirsutum cellulose synthase catalytic subunit 4 promoter in transgenic Arabidopsis and cotton tissues.

USDA-ARS?s Scientific Manuscript database

Gossypium hirsutum cellulose synthase catalytic subunit 4 (GhCesA4) plays an important role in cellulose biosynthesis during cotton fiber development. The transcript levels of GhCesA4 are significantly up-regulated as secondary cell wall cellulose is produced in developing cotton fibers. To unders...

DNA polymerase gamma from Xenopus laevis. I. The identification of a high molecular weight catalytic subunit by a novel DNA polymerase photolabeling procedure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Insdorf, N.F.; Bogenhagen, D.F.

1989-12-25

DNA polymerase gamma has been purified over 10,000-fold from mitochondria of Xenopus laevis ovaries. We have developed a novel technique which specifically photolabels DNA polymerases. This procedure, the DNA polymerase trap, was used to identify a catalytic subunit of 140,000 Da from X. laevis DNA polymerase gamma. Additional catalytically active polypeptides of 100,000 and 55,000 Da were identified in the highly purified enzyme. These appear to be products of degradation of the 140,000-Da subunit. The DNA polymerase trap, which does not require large amounts of enzyme or renaturation from sodium dodecyl sulfate, is an alternative to the classic activity gel.

Reverse-phase HPLC analysis of human alpha crystallin.

PubMed

Swamy, M S; Abraham, E C

1991-03-01

A rapid and highly sensitive reverse-phase HPLC (RP-HPLC) method was used to separate crystallin subunits from human alpha crystallin. Three distinct peaks were separated; by electrophoretic and immunological analyses the first and second peaks were identified as alpha B and alpha A respectively. On the other hand, peak 3 appeared to be a modified form of alpha crystallin. The ratio of alpha A and alpha B proteins was 3:1 in 1 day old lenses which gradually changed to 2:1 in 17 year old lenses and to 1:1 in the 50 and 82 year old whole lenses and 82 year old lens cortex, with a concomitant increase in the modified alpha, suggesting that alpha A subunits are relatively more involved in aggregation. Analysis of the 82 year old lens nucleus also supported this conclusion. The RP-HPLC analysis of the HMW aggregate fraction showed substantial enrichment of the modified alpha. The alpha A and alpha B subunits independently reassociated to form polymeric alpha crystallin whereas the modified alpha reassociated to form HMW aggregates as shown by molecular sieve HPLC. Hence it appears that the HMW aggregate peak was constituted by modified alpha crystallin. Only in the peak 3 material the 280 nm absorbance was about 2-fold higher than what was expected from the actual protein content. The data suggest that the changes induced by post-translational modifications may have some role in the formation of modified alpha. The present RP-HPLC method is useful in separating these modified alpha from the unmodified alpha A and alpha B subunits.

Calmodulin-dependent gating of Ca(v)1.2 calcium channels in the absence of Ca(v)beta subunits.

PubMed

Ravindran, Arippa; Lao, Qi Zong; Harry, Jo Beth; Abrahimi, Parwiz; Kobrinsky, Evgeny; Soldatov, Nikolai M

2008-06-10

It is generally accepted that to generate calcium currents in response to depolarization, Ca(v)1.2 calcium channels require association of the pore-forming alpha(1C) subunit with accessory Ca(v)beta and alpha(2)delta subunits. A single calmodulin (CaM) molecule is tethered to the C-terminal alpha(1C)-LA/IQ region and mediates Ca2+-dependent inactivation of the channel. Ca(v)beta subunits are stably associated with the alpha(1C)-interaction domain site of the cytoplasmic linker between internal repeats I and II and also interact dynamically, in a Ca2+-dependent manner, with the alpha(1C)-IQ region. Here, we describe a surprising discovery that coexpression of exogenous CaM (CaM(ex)) with alpha(1C)/alpha(2)delta in COS1 cells in the absence of Ca(v)beta subunits stimulates the plasma membrane targeting of alpha(1C), facilitates calcium channel gating, and supports Ca2+-dependent inactivation. Neither real-time PCR with primers complementary to monkey Ca(v)beta subunits nor coimmunoprecipitation analysis with exogenous alpha(1C) revealed an induction of endogenous Ca(v)beta subunits that could be linked to the effect of CaM(ex). Coexpression of a calcium-insensitive CaM mutant CaM(1234) also facilitated gating of Ca(v)beta-free Ca(v)1.2 channels but did not support Ca2+-dependent inactivation. Our results show there is a functional matchup between CaM(ex) and Ca(v)beta subunits that, in the absence of Ca(v)beta, renders Ca2+ channel gating facilitated by CaM molecules other than the one tethered to LA/IQ to support Ca2+-dependent inactivation. Thus, coexpression of CaM(ex) creates conditions when the channel gating, voltage- and Ca2+-dependent inactivation, and plasma-membrane targeting occur in the absence of Ca(v)beta. We suggest that CaM(ex) affects specific Ca(v)beta-free conformations of the channel that are not available to endogenous CaM.

Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

PubMed

Maurel, C; Kado, R T; Guern, J; Chrispeels, M J

1995-07-03

The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS)

Atomic force microscopy of ionotropic receptors bearing subunit-specific tags provides a method for determining receptor architecture

NASA Astrophysics Data System (ADS)

Neish, Calum S.; Martin, Ian L.; Davies, Martin; Henderson, Robert M.; Edwardson, J. Michael

2003-08-01

We have developed an atomic force microscopy (AFM)-based method for the determination of the subunit architecture of ionotropic receptors, and tested the method using the GABAA receptor as a model system. The most common form of the GABAA receptor probably consists of 2alpha1-, 2beta2- and 1gamma2-subunits. We show here that the arrangement of subunits around the central Cl- ion channel can be deduced by AFM of receptors tagged with subunit-specific antibodies. Transfection of cells with DNA encoding alpha1-, beta2- and gamma2-subunits resulted in the production of receptors containing all three subunits, as judged by both immunoblot analysis and the binding of [3H]-Ro15-1788, a specific radioligand for the GABAA receptor. A His6-tag on the alpha1-subunit was used to purify the receptor from membrane fractions of transfected cells. After incubation with anti-His6 immunoglobulin G, some receptors became tagged with either one or two antibody molecules. AFM analysis of complexes containing two bound antibodies showed that the most common angle between the two tags was 135°, close to the value of 144° expected if the two alpha-subunits are separated by a third subunit. This method is applicable to the complete elucidation of the subunit arrangement around the GABAA receptor rosette, and can also be applied to other ionotropic receptors.

Functional diversification of maize RNA polymerase IV and V subtypes via alternative catalytic subunits

DOE PAGES

Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; ...

2014-10-02

Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yieldsmore » at least two sub-types of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.« less

Functional diversification of maize RNA polymerase IV and V subtypes via alternative catalytic subunits

PubMed Central

Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; Sidorenko, Lyudmila; Nicora, Carrie D.; Norbeck, Angela D.; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; McGinnis, Karen; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

2014-01-01

Summary Unlike nuclear multisubunit RNA polymerases I, II and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA Polymerases IV and V are non-essential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their twelve subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits, but differ from each other only in their largest subunits. Use of alternative catalytic second-subunits, which are non-redundant for development and paramutation, yields at least two subtypes of Pol IV, and three subtypes of Pol V in maize. Pol IV/V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis. PMID:25284785

A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

PubMed

Xu, Shuhua; Soroka, Carol J; Sun, An-Qiang; Backos, Donald S; Mennone, Albert; Suchy, Frederick J; Boyer, James L

2016-01-01

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

PKA modulation of Kv4.2-encoded A-type potassium channels requires formation of a supramolecular complex.

PubMed

Schrader, Laura A; Anderson, Anne E; Mayne, Amber; Pfaffinger, Paul J; Sweatt, John David

2002-12-01

A-type channels, encoded by the pore-forming alpha-subunits of the Kv4.x family, are particularly important in regulating membrane excitability in the CNS and the heart. Given the key role of modulation of A currents by kinases, we sought to investigate the protein structure-function relationships underlying the regulation of these currents by PKA. We have previously shown the existence of two PKA phosphorylation sites in the Kv4.2 sequence; therefore, we focused this study on the Kv4.2 primary subunit. In the present studies we made the surprising finding that PKA phosphorylation of the Kv4.2 alpha-subunit is necessary but not sufficient for channel modulation; channel modulation by PKA required the presence of an ancillary subunit, the K+ channel interacting protein (KChIP3). Therefore, these findings indicate a surprising complexity to kinase regulation of A currents, in that an interaction of two separate molecular events, alpha-subunit phosphorylation and the association of an ancillary subunit (KChIP3), are necessary for phosphorylation-dependent regulation of Kv4.2-encoded A channels by PKA. Overall, our studies indicate that PKA must of necessity act on a supramolecular complex of pore-forming alpha-subunits plus ancillary subunits to alter channel properties.

Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum

PubMed Central

Ferguson, Scott A.; Cook, Gregory M.; Montgomery, Martin G.; Leslie, Andrew G. W.

2016-01-01

The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a “down” state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an “up” state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme’s hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual “open” conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis. PMID:27621435

Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit.

PubMed

Rettig, J; Heinemann, S H; Wunder, F; Lorra, C; Parcej, D N; Dolly, J O; Pongs, O

1994-05-26

Structural and functional diversity of voltage-gated Kv1-type potassium channels in rat brain is enhanced by the association of two different types of subunits, the membrane-bound, poreforming alpha-subunits and a peripheral beta-subunit. We have cloned a beta-subunit (Kv beta 1) that is specifically expressed in the rat nervous system. Association of Kv beta 1 with alpha-subunits confers rapid A-type inactivation on non-inactivating Kv1 channels (delayed rectifiers) in expression systems in vitro. This effect is mediated by an inactivating ball domain in the Kv beta 1 amino terminus.

PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis.

PubMed

Grinthal, Alison; Adamovic, Ivana; Weiner, Beth; Karplus, Martin; Kleckner, Nancy

2010-02-09

PR65 is the two-layered (alpha-alpha solenoid) HEAT-repeat (Huntingtin, elongation factor 3, a subunit of protein phosphatase 2A, PI3 kinase target of rapamycin 1) scaffold of protein phosphatase PP2A. Molecular dynamics simulations predict that, at forces expected in living systems, PR65 undergoes (visco-)elastic deformations in response to pulling/pushing on its ends. At lower forces, smooth global flexural and torsional changes occur via even redistribution of stress along the hydrophobic core of the molecule. At intermediate forces, helix-helix separation along one layer ("fracturing") leads to global relaxation plus loss of contact in the other layer to unstack the affected units. Fracture sites are determined by unusual sequences in contiguous interhelix turns. Normal mode analysis of the heterotrimeric PP2A enzyme reveals that its ambient conformational fluctuations are dominated by elastic deformations of PR65, which introduce a mechanical linkage between the separately bound regulatory and catalytic subunits. PR65-dominated fluctuations of PP2A have the effect of opening and closing the enzyme's substrate binding/catalysis interface, as well as altering the positions of certain catalytic residues. These results suggest that substrate binding/catalysis are sensitive to mechanical force. Force could be imposed from the outside (e.g., in PP2A's response to spindle tension) or arise spontaneously (e.g., in PP2A's interaction with unstructured proteins such as Tau, a microtubule-associated Alzheimer's-implicated protein). The presented example supports the view that conformation and function of protein complexes can be modulated by mechanical energy inputs, as well as by chemical energy inputs from ligand binding. Given that helical-repeat proteins are involved in many cellular processes, the findings also encourage the view that mechanical forces may be of widespread importance.

Gα proteins Gvm2 and Gvm3 regulate vegetative growth, asexual development, and pathogenicityon apple in Valsa mali.

PubMed

Song, Na; Dai, Qingqing; Zhu, Baitao; Wu, Yuxing; Xu, Ming; Voegele, Ralf Thomas; Gao, Xiaoning; Kang, Zhensheng; Huang, Lili

2017-01-01

In fungi, heterotrimeric guanine-nucleotide binding proteins (G-proteins) are key elements of signal transduction pathways, which control growth, asexual and sexual development, as well as virulence. In this study, we have identified two genes encoding heterotrimeric G protein alpha subunits, named Gvm2 and Gvm3, from Valsa mali, the causal agent of apple Valsa canker. Characterization of Gvm2 and Gvm3 mutants indicates that Gvm3 may be a crucial regulator of vegetative growth. Deletion of the corresponding gene results in a 20% reduction in growth rate. Besides, Gvm2 and Gvm3 seem to be involved in asexual reproduction, and mutants are hypersensitive to oxidative and cell membrane stresses. Interestingly, both G protein alpha subunits were most probably involved in V. mali virulence. In infection assays using Malus domestica cv. 'Fuji' leaves and twigs, the size of lesions caused by deletion mutants △Gvm2, or △Gvm3 are significantly reduced. Furthermore, many genes encoding hydrolytic enzymes-important virulence factors in V. mali-are expressed at a lower level in these deletion mutants. Our results suggest that Gvm2 and Gvm3 play an important role in virulence probably by regulation of expression of cell wall degrading enzymes. △Gvm2, and △Gvm3 mutants were further analyzed with respect to their impact on the transcript levels of genes in the cAMP/PKA pathway. The expression of the genes encoding adenylate cyclase VmAC, protein kinase A (PKA) regulatory subunit VmPKR, and PKA catalytic subunit VmPKA1 are down-regulated in both mutants. Further analyses indicated that intracellular cAMP level and PKA activity are down-regulated in the △Gvm3 mutant, but are basically unchanged in the △Gvm2 mutant. Overall, our findings indicate that both Gvm2 and Gvm3 play diverse roles in the modulation of vegetative growth, asexual development, and virulence in V. mali.

Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography.

PubMed

Sauguet, Ludovic; Raia, Pierre; Henneke, Ghislaine; Delarue, Marc

2016-08-22

Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same 'double-psi β-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs.

Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography

PubMed Central

Sauguet, Ludovic; Raia, Pierre; Henneke, Ghislaine; Delarue, Marc

2016-01-01

Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same ‘double-psi β-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs. PMID:27548043

Ferritins: dynamic management of biological iron and oxygen chemistry.

PubMed

Liu, Xiaofeng; Theil, Elizabeth C

2005-03-01

Ferritins are spherical, cage-like proteins with nanocavities formed by multiple polypeptide subunits (four-helix bundles) that manage iron/oxygen chemistry. Catalytic coupling yields diferric oxo/hydroxo complexes at ferroxidase sites in maxi-ferritin subunits (24 subunits, 480 kDa; plants, animals, microorganisms). Oxidation occurs at the cavity surface of mini-ferritins/Dps proteins (12 subunits, 240 kDa; bacteria). Oxidation products are concentrated as minerals in the nanocavity for iron-protein cofactor synthesis (maxi-ferritins) or DNA protection (mini-ferritins). The protein cage and nanocavity characterize all ferritins, although amino acid sequences diverge, especially in bacteria. Catalytic oxidation/di-iron coupling in the protein cage (maxi-ferritins, 480 kDa; plants, bacteria and animal cell-specific isoforms) or on the cavity surface (mini-ferritins/Dps proteins, 280 kDa; bacteria) initiates mineralization. Gated pores (eight or four), symmetrically arranged, control iron flow. The multiple ferritin functions combine pore, channel, and catalytic functions in compact protein structures required for life and disease response.

The sodium pump alpha1 subunit as a potential target to combat apoptosis-resistant glioblastomas.

PubMed

Lefranc, Florence; Kiss, Robert

2008-03-01

To review the involvement of the ion transporter Na+/K+-ATPase (NaK) in the migration and proliferation of glioma cells. Preliminary studies indicate that NaK alpha1 subunits seem to be upregulated in a proportion of glioblastomas but not in normal brain tissues. The present review focuses on (1) the natural resistance of migrating malignant glioma cells to apoptosis, (2) autophagic cell death as an alternative to combat malignant gliomas, (3) the fact that reducing the levels of malignant glioma cell motility can restore proapoptotic drug sensitivity,and (4) on the observation that inhibiting the NaK activity reduces both glioma cell proliferation and migration. The natural ligands of the NaK are the cardiotonic steroids. A hemisynthetic derivative of 2"-oxovoruscharin (UNBS1450), a novel cardenolide, displays unique structural features, making its binding affinity to NaK alpha subunits (including alpha1) 10 to 100 times higher than that of other cardenolides. UNBS1450 markedly decreases intracellular ATP concentration in glioma cells, disorganizes the actin cytoskeleton, and leads to autophagic cell death in NaK alpha1 over-expressing glioma cells. Glioblastoma patients who do not respond to chemotherapy and whose tumors over-express NaK alpha1 subunits could benefit from a treatment using ligands with marked binding affinity for the NaK alpha1 subunit.

Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCormick, D.J.; Griesmann, G.E.; Huang, Z.

1986-03-05

A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodiesmore » to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.« less

Protein Kinase A Subunit Balance Regulates Lipid Metabolism in Caenorhabditis elegans and Mammalian Adipocytes*

PubMed Central

Lee, Jung Hyun; Han, Ji Seul; Kong, Jinuk; Ji, Yul; Lv, Xuchao; Lee, Junho; Li, Peng; Kim, Jae Bum

2016-01-01

Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RIα and RIIβ via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism. PMID:27496951

Neuroblastoma differentiation involves the expression of two isoforms of the alpha-subunit of Go.

PubMed

Brabet, P; Pantaloni, C; Rodriguez, M; Martinez, J; Bockaert, J; Homburger, V

1990-04-01

The regulation of GTP-binding proteins (G proteins) was examined during the course of differentiation of neuroblastoma N1E-115 cells. N1E-115 cell membranes possess three Bordetella pertussis toxin (PTX) substrates assigned to alpha-subunits (G alpha) of Go (a G protein of unknown function) and "Gi (a G protein inhibitory to adenylate cyclase)-like" proteins and one substrate of Vibrio cholerae toxin corresponding to an alpha-subunit of Gs (a G protein stimulatory to adenylate cyclase). In undifferentiated cells, only one form of Go alpha was found, having a pI of 5.8 Go alpha content increased by approximately twofold from the undifferentiated state to 96 h of cell differentiation. This is mainly due to the appearance of another Go alpha form having a pI of 5.55. Both Go alpha isoforms have similar sizes on sodium dodecyl sulfate-polyacrylamide gels, are recognized by polyclonal antibodies to bovine brain Go alpha, are ADP-ribosylated by PTX, and are covalently myristylated in whole N1E-115 cells. In addition, immunofluorescent staining of N1E-115 cells with Go alpha antibodies revealed that association of Go alpha with the plasma membrane appears to coincide with the expression of the most acidic isoform and morphological cell differentiation. In contrast, the levels of both Gi alpha and Gs alpha did not significantly change, whereas that of the common beta-subunit increased by approximately 30% over the same period. These results demonstrate specific regulation of the expression of Go alpha during neuronal differentiation.

A cross-linking study of the Ca2+, Mg2+-activated adenosine triphosphatase of Escherichia coli.

PubMed

Bragg, P D; Hou, C

1980-05-01

The solubilized Ca2+,Mg2+-activated adenosine triphosphatase of Escherichia coli is composed of five subunits designated alpha, beta, gamma, delta and epsilon in order of decreasing molecular weight. The subunit structure of the enzyme has been investigated by the use of the cleavable cross-linking agents dithiobis(succinimidyl propionate), methyl-4-mercaptobutyrimidate, dimethyl-3,3'-dithiobispropionimidate, disuccinimidyl tartarate, and cupric 1,10-phenanthrolinate. The products of cross-linking were analyzed by two different two-dimensional gel electrophoresis systems. The following cross-linked subunit dimers were observed: alpha 2, beta 2, alpha beta, alpha delta, beta gamma, beta delta, beta epsilon and gamma epsilon. These results, together with other published data, are discussed in relation to a model of the arrangement of the subunits in the ATPase molecule.

Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development.

PubMed

Beier, Anna; Teichert, Ines; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

2016-06-21

The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood. The first fungal STRIPAK was described in Sordaria macrospora, which is a well-established model organism used to study the formation of fungal fruiting bodies, three-dimensional organ-like structures. We analyzed STRIPAK subunit PP2Ac1, catalytic subunit 1 of protein phosphatase PP2A, to study the importance of the catalytic activity of this protein during sexual development. The results of our yeast two-hybrid analysis and tandem affinity purification, followed by mass spectrometry, indicate that PP2Ac1 activity connects STRIPAK with other signaling pathways and thus forms a large interconnected signaling network. Copyright © 2016 Beier et al.

Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kapoor, Neeraj; Menon, Santosh T.; Chauhan, Radha

2010-01-12

Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5'-diphosphate (GDP) release by the G protein {alpha}-subunit is not well understood. Amino acid substitutions in the conserved {alpha}5 helix of Gi, which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant G{alpha}{sub i1}-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of G{alpha}{submore » i1}-T329A {center_dot} GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg{sup +2} coordination sphere and dislodge bound Mg{sup +2}. We propose a 'sequential release' mechanism whereby a transient conformational change in the {alpha}5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.« less

Computer modeling of siRNA knockdown effects indicates an essential role of the Ca2+ channel alpha2delta-1 subunit in cardiac excitation-contraction coupling.

PubMed

Tuluc, Petronel; Kern, Georg; Obermair, Gerald J; Flucher, Bernhard E

2007-06-26

L-type Ca(2+) currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca(2+) signal, which regulates the contraction force. The auxiliary Ca(2+) channel subunits alpha(2)delta-1 and beta(2) are important regulators of membrane expression and current properties of the cardiac Ca(2+) channel (Ca(V)1.2). However, their role in cardiac excitation-contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the alpha(2)delta-1 subunit in a muscle expression system with simulation of APs and Ca(2+) transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with Ca(V)1.2 (GFP-alpha(1C)) recapitulates key properties of cardiac excitation-contraction coupling. Concomitant depletion of the alpha(2)delta-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-alpha(1C) subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, alpha(2)delta-1 depletion shifted the voltage dependence of Ca(2+) current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic Ca(2+) concentration during each contraction. Thus, the Ca(2+) channel alpha(2)delta-1 subunit is not essential for normal Ca(2+) channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of alpha(2)delta-1 function is predicted to severely perturb normal heart function.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spreitzer, Robert J.

CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesismore » is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.« less

A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins

PubMed Central

1995-01-01

To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin. PMID:7721947

Structural Model for the Flip-Flop Action in Thiamin Pyrophosphate-Dependent Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Dominiak, Paulina

2003-01-01

The derivative of vitamin B1 thiamin pyrophosphate (TPP) is a cofactor of enzymes performing catalysis in pathways of energy production, including (i) decarboxylation of alpha-keto acids followed by (ii) transketolation. These enzymes have shown a common mechanism of TPP activation by imposing an active V-conformation of this coenzyme that brings the N4 atom of the aminopyrimidine ring to the distance required for the intramolecular C-H N hydrogen-bonding with the C2- atom of the thiazolium ring. The reactive C2 atom of TPP is the nucleophile that attacks the carbonyl carbon of different substrates used by the TPP-dependent enzymes. The structure of the heterotetrameric human pyruvate dehydrogenase (Elp) recently determined in our laboratory (1) revealed the association pattern of the subunits and the specifics of two chemically equivalent cofactor binding sites. Dynamic nonequivalence of these two cofactor sites directs the flip-flop action of this enzyme, depending upon which two active sites effect each other (2). The crystal structure derived from the holo-form of Elp provided the basis for the model of the flip-flop action of Elp in which different steps of the catalytic reaction are performed in each of the two cofactor sites at any given moment, where these steps are governed by the concerted shuttle-like motion of the subunits. It is further proposed that balancing a hydrogen-bond network and related cofactor geometry determine the continuity of catalytic events.

Subunit assembly of hemoglobin: an important determinant of hematologic phenotype.

PubMed

Bunn, H F

1987-01-01

Hemoglobin's physiologic properties depend on the orderly assembly of its subunits in erythropoietic cells. The biosynthesis of alpha- and beta-globin polypeptide chains is normally balanced. Heme rapidly binds to the globin subunit, either during translation or shortly thereafter. The formation of the alpha beta-dimer is facilitated by electrostatic attraction of a positively charged alpha-subunit to a negatively charged beta-subunit. The alpha beta-dimer dissociates extremely slowly. The difference between the rate of dissociation of alpha beta- and alpha gamma-dimers with increasing pH explains the well-known alkaline resistance of Hb F. Two dimers combine to form the functioning alpha 2 beta 2-tetramer. This model of hemoglobin assembly explains the different levels of positively charged and negatively charged mutant hemoglobins that are encountered in heterozygotes and the effect of alpha-thalassemia and heme deficiency states in modifying the level of the variant hemoglobin as well as Hb A2. Electrostatic interactions also affect the binding of hemoglobin to the cytoplasmic surface of the red cell membrane and may underlie the formation of target cells. Enhanced binding of positively charged variants such as S and C trigger a normally dormant pathway for potassium and water loss. Thus, the positive charge on beta c is responsible for the two major contributors to the pathogenesis of Hb SC disease: increased proportion of Hb S and increased intracellular hemoglobin concentration. It is likely that electrostatic interactions play an important role in the assembly of a number of other multisubunit macromolecules, including membrane receptors, cytoskeletal proteins, and DNA binding proteins.

The primary and subunit structure of a novel type killer toxin produced by a halotolerant yeast, Pichia farinosa.

PubMed

Suzuki, C; Nikkuni, S

1994-01-28

A halotolerant yeast, Pichia farinosa KK1 strain, produces a unique killer toxin termed SMK toxin (salt-mediated killer toxin) which shows its maximum killer activity in the presence of 2 M NaCl. The toxin consists of two distinct subunits, alpha and beta, which are tightly linked without a disulfide bond under acidic conditions, even in the presence of 6 M urea. Under neutral conditions, however, the alpha subunit precipitates, resulting in the dissociation of the subunits and the loss of killer activity. The nucleotide sequence of the SMK1 gene predicts a 222 amino acid preprotoxin with a typical signal sequence, the hydrophobic alpha, an interstitial gamma polypeptide with a putative glycosylation site, and the hydrophilic beta. Amino acid sequence analyses of peptide fragments including the carboxyl-terminal peptides fragments including the carboxyl-terminal peptides from each subunit suggest that the alpha and beta subunits consist of amino acid residues 19-81 and 146-222 of the preprotoxin, respectively, and the molecular weight of the mature alpha beta dimer is 14,214. The KEX2-like endopeptidase and KEX1-like carboxypeptidase may be involved in the stepwise processing of the SMK preprotoxin. The maturation process and the functions of the SMK toxin are compared with the K1 toxin of Saccharomyces cerevisiae.

Homologous kappa-neurotoxins exhibit residue-specific interactions with the alpha 3 subunit of the nicotinic acetylcholine receptor: a comparison of the structural requirements for kappa-bungarotoxin and kappa-flavitoxin binding.

PubMed

McLane, K E; Weaver, W R; Lei, S; Chiappinelli, V A; Conti-Tronconi, B M

1993-07-13

kappa-Flavotoxin (kappa-FTX), a snake neurotoxin that is a selective antagonist of certain neuronal nicotinic acetylcholine receptors (AChRs), has recently been isolated and characterized [Grant, G. A., Frazier, M. W., & Chiappinelli, V. A. (1988) Biochemistry 27, 1532-1537]. Like the related snake toxin kappa-bungarotoxin (kappa-BTX), kappa-FTX binds with high affinity to alpha 3 subtypes of neuronal AChRs, even though there are distinct sequence differences between the two toxins. To further characterize the sequence regions of the neuronal AChR alpha 3 subunit involved in formation of the binding site for this family of kappa-neurotoxins, we investigated kappa-FTX binding to overlapping synthetic peptides screening the alpha 3 subunit sequence. A sequence region forming a "prototope" for kappa-FTX was identified within residues alpha 3 (51-70), confirming the suggestions of previous studies on the binding of kappa-BTX to the alpha 3 subunit [McLane, K. E., Tang, F., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 1537-1544] and alpha-bungarotoxin to the Torpedo AChR alpha subunit [Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230] that this sequence region is involved in formation of a cholinergic site. Single residue substituted analogues, where each residue of the sequence alpha 3 (51-70) was sequentially replaced by a glycine, were used to identify the amino acid side chains involved in the interaction of this prototope with kappa-FTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the mechanism of activation of cAMP-dependent protein kinase through the study of mutants of the yeast regulatory subunit.

PubMed

Zaremberg, V; Moreno, S

1996-04-01

Spontaneous mutations in the gene which encodes the regulatory subunit of cAMP-dependent protein kinase (PKA) of Saccharomyces cerevisiae (BCY1) have been isolated previously [Cannon, J. F., Gibbs, J. B. & Tatchell, K. (1986) Genetics 113, 247-264] by selection of ras2::LEU2 revertants that grew on non-fermentable carbon sources. The revertants were placed into groups of increasing severity based on the number of PKA-dependent traits affected [Cannon, J. F., Gitan, R. & Tatchell, K. (1990) J. Biol. Chem. 265, 11897-11904]. In this work the ras2 mutation has been crossed out in each bcy1 allele and the phenotypes of these mutants have been assessed. The order of severity of the mutants in both genetic backgrounds is maintained but the severity of each mutant in the normal background is higher than in the ras2::LEU2 background. Total catalytic-subunit and regulatory-subunit activities were measured in crude extracts of the bcy1 ras2::LEU2 mutants. With one exception (bcy1-6) the calculated regulatory subunit/catalytic subunit ratios of the bcy1 mutants relative to that of wild-type cells were greater than one. The dependence of PKA activity on cAMP was measured in permeabilized cells. The strains show an activity ratio in the absence and presence of cAMP in the range 0.5-1 for Kemptide phosphorylation. Overexpression of the high-affinity cAMP phosphodiesterase gene (PDE2) in the bcy1 ras2::LEU2 strains did not alter their PKA-dependent phenotypes. However, transformants were not observed from the parental ras2::LEU2 strain and the bcy1-6 ras2::LEU2 strain. The results are discussed with respect to a hypothesis for the molecular mechanism of the differential reversal of ras2 phenotypes by the bcy1 alleles. Mutations in the regulatory subunit are predicted to affect the structure of the holoenzyme such that the catalytic subunit is capable of maintaining an active catalytic state, without the need to dissociate from the regulatory subunit.

Expression of the alpha and beta subunits of Ca2+/calmodulin kinase II in the cerebellum of jaundiced Gunn rats during development: a quantitative light microscopic analysis.

PubMed

Conlee, J W; Shapiro, S M; Churn, S B

2000-04-01

The homozygous (jj) jaundiced Gunn rat model for hyperbilirubinemia displays pronounced cerebellar hypoplasia. To examine the cellular mechanisms involved in bilirubin toxicity, this study focused on the effect of hyperbilirubinemia on calcium/calmodulin-dependent kinase II (CaM kinase II). CaM kinase II is a neuronally enriched enzyme which performs several important functions. Immunohistochemical analysis of alternating serial sections were performed using monoclonal antibodies for the alpha and beta subunits of CaM kinase II. Measurements were made of the total numbers of stained cells in each of the deep cerebellar nuclei and of Purkinje and granule cell densities in cerebellar lobules II, VI, and IX. The beta subunit was present in Purkinje cells and deep cerebellar nuclei of both groups at all ages, but only granule cells which had migrated through the Purkinje cell layer showed staining for beta subunit; external granule cells were completely negative. Many Purkinje cells had degenerated in the older animals, and the percent of granule cells stained for beta subunit was significantly reduced. The alpha subunit was found exclusively in Purkinje cells, although its appearance was delayed in the jaundiced animals. Sulfadimethoxine was administered to some jj rats 24 h or 15 days prior to sacrifice to increase brain bilirubin concentration. Results showed that bilirubin exposure modulated both alpha and beta CaM kinase II subunit expression in selective neuronal populations, but sulfadimethoxine had no acute effect on enzyme immunoreactivity. Thus, developmental expression of the alpha and beta subunits of CaM kinase II was affected by chronic bilirubin exposure during early postnatal development of jaundiced Gunn rats.

Molecular analysis of nicotinic receptor expression in autism.

PubMed

Martin-Ruiz, C M; Lee, M; Perry, R H; Baumann, M; Court, J A; Perry, E K

2004-04-07

Autism is a developmental disorder of unknown aetiopathology and lacking any specific pharmacological therapeutic intervention. Neurotransmitters such as serotonin, gamma-aminobutyric acid (GABA) and acetylcholine have been implicated. Abnormalities in nicotinic acetylcholine receptors have been identified including cortical loss of binding to the alpha4/beta2 subtype and increase in cerebellar alpha7 binding. Receptor expression (mRNA) has not so far been systematically examined. This study aims to further explore the role of nicotinic receptors in autism by analysing nicotinic receptor subunit mRNA in conjunction with protein levels and receptor binding in different brain areas. Quantitative RT-PCR for alpha4, alpha7 and beta2 subunit mRNA expression levels; alpha3, alpha4, alpha7 and beta2 subunit protein expression immunochemistry and specific radioligand receptor binding were performed in adult autism and control brain samples from cerebral cortex and cerebellum. Alpha4 and beta2 protein expression and receptor binding density as well as alpha4 mRNA levels were lower in parietal cortex in autism, while alpha7 did not change for any of these parameters. In cerebellum, alpha4 mRNA expression was increased, whereas subunit protein and receptor levels were decreased. Alpha7 receptor binding in cerebellum was increased alongside non-significant elevations in mRNA and protein expression levels. No significant changes were found for beta2 in cerebellum. The data obtained, using complementary measures of receptor expression, indicate that reduced gene expression of the alpha4beta2 nicotinic receptor in the cerebral cortex is a major feature of the neurochemical pathology of autism, whilst post-transcriptional abnormalities of both this and the alpha7 subtype are apparent in the cerebellum. The findings point to dendritic and/or synaptic nicotinic receptor abnormalities that may relate to disruptions in cerebral circuitry development.

Catalytic asymmetric epoxidation of alpha,beta-unsaturated amides: efficient synthesis of beta-aryl alpha-hydroxy amides using a one-pot tandem catalytic asymmetric epoxidation-Pd-catalyzed epoxide opening process.

PubMed

Nemoto, Tetsuhiro; Kakei, Hiroyuki; Gnanadesikan, Vijay; Tosaki, Shin-Ya; Ohshima, Takashi; Shibasaki, Masakatsu

2002-12-11

The catalytic asymmetric epoxidation of alpha,beta-unsaturated amides using Sm-BINOL-Ph3As=O complex was succeeded. Using 5-10 mol % of the asymmetric catalyst, a variety of amides were epoxidized efficiently, yielding the corresponding alpha,beta-epoxy amides in up to 99% yield and in more than 99% ee. Moreover, the novel one-pot tandem process, one-pot tandem catalytic asymmetric epoxidation-Pd-catalyzed epoxide opening process, was developed. This method was successfully utilized for the efficient synthesis of beta-aryl alpha-hydroxy amides, including beta-aryllactyl-leucine methyl esters. Interestingly, it was found that beneficial modifications on the Pd catalyst were achieved by the constituents of the first epoxidation, producing a more suitable catalyst for the Pd-catalyzed epoxide opening reaction in terms of chemoselectivity.

A saposin-like domain influences the intracellular localization, stability, and catalytic activity of human acyloxyacyl hydrolase.

PubMed

Staab, J F; Ginkel, D L; Rosenberg, G B; Munford, R S

1994-09-23

Acyloxyacyl hydrolase, a leukocyte enzyme that acts on bacterial lipopolysaccharides (LPSs) and many glycerolipids, is synthesized as a precursor polypeptide that undergoes internal disulfide linkage before being proteolytically processed into two subunits. The larger subunit contains an amino acid sequence (Gly-X-Ser-X-Gly) that is found at the active sites of many lipases, while the smaller subunit has amino acid sequence similarity to saposins (sphingolipid activator proteins), cofactors for sphingolipid glycohydrolases. We show here that both acyloxyacyl hydrolase subunits are required for catalytic activity toward LPS and glycerophosphatidylcholine. In addition, mutations that truncate or delete the small subunit have profound effects on the intracellular localization, proteolytic processing, and stability of the enzyme in baby hamster kidney cells. Remarkably, proteolytic cleavage of the precursor protein increases the activity of the enzyme toward LPS by 10-20-fold without altering its activity toward glycerophosphatidylcholine. Proper orientation of the two subunits thus seems very important for the substrate specificity of this unusual enzyme.

Exercise increases the plasma membrane content of the Na+ -K+ pump and its mRNA in rat skeletal muscles.

PubMed

Tsakiridis, T; Wong, P P; Liu, Z; Rodgers, C D; Vranic, M; Klip, A

1996-02-01

Muscle fibers adapt to ionic challenges of exercise by increasing the plasma membrane Na+-K+ pump activity. Chronic exercise training has been shown to increase the total amount of Na+-K+ pumps present in skeletal muscle. However, the mechanism of adaptation of the Na+-K+ pump to an acute bout of exercise has not been determined, and it is not known whether it involves alterations in the content of plasma membrane pump subunits. Here we examine the effect of 1 h of treadmill running (20 m/min, 10% grade) on the subcellular distribution and expression of Na+-K+ pump subunits in rat skeletal muscles. Red type I and IIa (red-I/IIa) and white type IIa and IIb (white-IIa/IIb) hindlimb muscles from resting and exercised female Sprague-Dawley rats were removed for subcellular fractionation. By homogenization and gradient centrifugation, crude membranes and purified plasma membranes were isolated and subjected to gel electrophoresis and immunoblotting by using pump subunit-specific antibodies. Furthermore, mRNA was isolated from specific red type I (red-I) and white type IIb (white-IIb) muscles and subjected to Northern blotting by using subunit-specific probes. In both red-I/IIa and white-IIa/IIb muscles, exercise significantly raised the plasma membrane content of the alpha1-subunit of the pump by 64 +/- 24 and 55 +/- 22%, respectively (P < 0.05), and elevated the alpha2-polypeptide by 43 +/- 22 and 94 +/- 39%, respectively (P < 0.05). No significant effect of exercise could be detected on the amount of these subunits in an internal membrane fraction or in total membranes. In addition, exercise significantly increased the alpha1-subunit mRNA in red-I muscle (by 50 +/- 7%; P < 0.05) and the beta2-subunit mRNA in white-IIb muscles (by 64 +/- 19%; P < 0.01), but the alpha2- and beta1-mRNA levels were unaffected in this time period. We conclude that increased presence of alpha1- and alpha2-polypeptides at the plasma membrane and subsequent elevation of the alpha1- and beta2-subunit mRNAs may be mechanisms by which acute exercise regulates the Na+-K+ pump of skeletal muscle.

An all sulfur analogue of the smallest subunit of F420-non-reducing hydrogenase from Methanococcus voltae--metal binding and structure.

PubMed

Pfeiffer, M; Klein, A; Steinert, P; Schomburg, D

The 25 amino acid long subunit VhuU of the F420-non-reducing hydrogenase from Methanococcus voltae contains selenocysteine within the consensus sequence of known [NiFe] hydrogenases DP(C or U)CxxCxxH (U = selenocysteine). The sulfur-analogue VhuUc was chemically synthesized, purified and its metal binding capability, the catalytic properties, and structural features were investigated. The polypeptide was able to bind nickel, but did not catalyse the heterolytic activation of H2. 2D-NMR spectroscopy revealed an alpha-helical secondary structure for the 15 N-terminal amino acids in 50% TFE. Nickel only binds to the C-terminus, which contains the conserved amino acid motif. Structures derived from the NMR data are compatible with the participation of both sulfur atoms from the conserved cysteine residues in a metal ion binding. Structures obtained from the data sets for Ni.VhuUc as well as Zn.VhuUc showed no further ligands. The informational value for Ni.VhuUc was low due to paramagnetism.

Chemical cross-linking of the urease complex from Helicobacter pylori and analysis by Fourier transform ion cyclotron resonance mass spectrometry and molecular modeling

NASA Astrophysics Data System (ADS)

Carlsohn, Elisabet; Ångström, Jonas; Emmett, Mark R.; Marshall, Alan G.; Nilsson, Carol L.

2004-05-01

Chemical cross-linking of proteins is a well-established method for structural mapping of small protein complexes. When combined with mass spectrometry, cross-linking can reveal protein topology and identify contact sites between the peptide surfaces. When applied to surface-exposed proteins from pathogenic organisms, the method can reveal structural details that are useful in vaccine design. In order to investigate the possibilities of applying cross-linking on larger protein complexes, we selected the urease enzyme from Helicobacter pylori as a model. This membrane-associated protein complex consists of two subunits: [alpha] (26.5 kDa) and [beta] (61.7 kDa). Three ([alpha][beta]) heterodimers form a trimeric ([alpha][beta])3 assembly which further associates into a unique dodecameric 1.1 MDa complex composed of four ([alpha][beta])3 units. Cross-linked peptides from trypsin-digested urease complex were analyzed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and molecular modeling. Two potential cross-linked peptides (present in the cross-linked sample but undetectable in [alpha], [beta], and native complex) were assigned. Molecular modeling of urease [alpha][beta] complex and trimeric urease units ([alpha][beta])3 revealed a linkage site between the [alpha]-subunit and the [beta]-subunit, and an internal cross-linkage in the [beta]-subunit.

[Tonoplast transport and salt tolerance in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taiz, L.

1993-01-01

We have showed that the tonoplast V-ATPase could be specifically inhibited by antisense DNA to the catalytic (A) subunit; that cell expansion was inhibited in carrot transformants deficient in the enzyme and have provided evidence for at least two different isoforms of the A subunit which are Golgi- and tonoplast-specific. These findings prompted a search for sequences of the isoforms of the A subunit in carrot. We have cloned and sequenced 1.0--1.5 kb fragments of three different genes for the catalytic subunit, the fragments differ greatly in their introns, but have nearly identical exons. We are using PCR to amplifymore » and subclone carrot seedling cDNA. Thus far two bands have been amplified and are currently being subcloned for sequencing.« less

[Tonoplast transport and salt tolerance in plants]. Progress report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taiz, L.

1993-04-01

We have showed that the tonoplast V-ATPase could be specifically inhibited by antisense DNA to the catalytic (A) subunit; that cell expansion was inhibited in carrot transformants deficient in the enzyme and have provided evidence for at least two different isoforms of the A subunit which are Golgi- and tonoplast-specific. These findings prompted a search for sequences of the isoforms of the A subunit in carrot. We have cloned and sequenced 1.0--1.5 kb fragments of three different genes for the catalytic subunit, the fragments differ greatly in their introns, but have nearly identical exons. We are using PCR to amplifymore » and subclone carrot seedling cDNA. Thus far two bands have been amplified and are currently being subcloned for sequencing.« less

A novel mutation in the alpha-helix 1 of the C subunit of the F(1)/F(0) ATPase responsible for optochin resistance of a Streptococcus pneumoniae clinical isolate.

PubMed

Cogné, N; Claverys, J; Denis, F; Martin, C

2000-10-01

Previously reported mutations involved in optochin resistance of Streptococcus pneumoniae clinical isolates changed residues 48, 49 or 50, in the transmembrane alpha-helix 2 of the F(1)/F(0) ATPase subunit. We report here an unusual mutation which changes the sequence of the transmembrane alpha-helix 1 of the AtpC subunit. This mutation involves a Gly to Ser substitution resulting from a G to A transition at codon 14 of the atpC gene.

Na(+)-K (+) pump location and translocation during muscle contraction in rat skeletal muscle.

PubMed

Kristensen, Michael; Rasmussen, Martin Krøyer; Juel, Carsten

2008-08-01

Muscle contraction may up-regulate the number of Na(+)-K(+) pumps in the plasma membrane by translocation of subunits. Since there is still controversy about where this translocation takes place from and if it takes place at all, the present study used different techniques to characterize the translocation. Electrical stimulation and biotin labeling of rat muscle revealed a 40% and 18% increase in the amounts of the Na(+)-K(+) pump alpha(2) subunit and caveolin-3 (Cav-3), respectively, in the sarcolemma. Exercise induced a 36% and 19% increase in the relative amounts of the alpha(2) subunit and Cav-3, respectively, in an outer-membrane-enriched fraction and a 41% and 17% increase, respectively, in sarcolemma giant vesicles. The Na(+)-K(+) pump activity measured with the 3-O-MFPase assay was increased by 37% in giant vesicles from exercised rats. Immunoprecipitation with Cav-3 antibody showed that 17%, 11% and 14% of the alpha(1) subunits were associated with Cav-3 in soleus, extensor digitorum longus, and mixed muscles, respectively. For the alpha(2), the corresponding values were 17%, 5% and 16%. In conclusion; muscle contraction induces translocation of the alpha subunits, which is suggested to be caused partly by structural changes in caveolae and partly by translocation from an intracellular pool.

Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact.

PubMed

Heyduk, E; Baichoo, N; Heyduk, T

2001-11-30

The alpha-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence. To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the alpha-subunit of E. coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization. The alpha dimer and DNA formed a 1:1 complex in solution. Complex formation at 25 degrees C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed. The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (Deltac(p)) was close to zero. Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation. No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation. The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the alpha-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between alpha-subunit and DNA was not formed.

Structure of the beta 2 homodimer of bacterial luciferase from Vibrio harveyi: X-ray analysis of a kinetic protein folding trap.

PubMed Central

Thoden, J. B.; Holden, H. M.; Fisher, A. J.; Sinclair, J. F.; Wesenberg, G.; Baldwin, T. O.; Rayment, I.

1997-01-01

Luciferase, as isolated from Vibrio harveyi, is an alpha beta heterodimer. When allowed to fold in the absence of the alpha subunit, either in vitro or in vivo, the beta subunit of enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 degrees C. This form of the beta subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1: 320-326). Here we describe the X-ray crystal structure of the beta 2 homodimer of luciferase from V. harveyi determined and refined at 1.95 A resolution. Crystals employed in the investigational belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 58.8 A, b = 62.0 A, and c = 218.2 A and contained one dimer per asymmetric unit. Like that observed in the functional luciferase alpha beta heterodimer, the major tertiary structural motif of each beta subunit consists of an (alpha/beta)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 A resolution. Biochemistry 34: 6581-6586). The root-mean-square deviation of the alpha-carbon coordinates between the beta subunits of the hetero- and homodimers is 0.7 A. This high resolution X-ray analysis demonstrated that "domain" or "loop" swapping has not occurred upon formation of the beta 2 homodimer and thus the stability of the beta 2 species to denaturation cannot be explained in such simple terms. In fact, the subunit:subunit interfaces observed in both the beta 2 homodimer and alpha beta heterodimer are remarkably similar in hydrogen-bonding patterns and buried surface areas. PMID:9007973

Phenotypic consequences of deletion of the {gamma}{sub 3}, {alpha}{sub 5}, or {beta}{sub 3} subunit of the type A {gamma}-aminobutyric acid receptor in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Culia, C.T.; Stubbs, L.J.; Montgomery, C.S.

1994-03-29

Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the {gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3} subunits of the type A {gamma}-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3-Gabra5-Gabrb3-telomere. Like Gabrb3, neither the Gabra5 nor Gabrg3 gene is functionally imprinted in adult mouse brain. Mice deleted for all three subunits die at birth with a cleft palate, although there are rare survivors ({approximately} 5%) that do notmore » have a cleft palate but do exhibit a neurological abnormality characterized by tremor, jerky gait, and runtiness. The authors have previously suggested that deficiency of the {beta}{sub 3} subunit may be responsible for the clefting defect. Most notably, however, in this report they describe mice carrying two overlapping, complementing p deletions that fail to express the {gamma}{sub 3} transcript, as well as mice from another line that express neither the {gamma}{sub 3} nor {alpha}{sub 5} transcripts. Surprisingly, mice from both of these lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three ({gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3}) subunits. These mice therefore provide a whole-organism type A {gamma}-aminobutyric-acid receptor background that is devoid of any receptor subtypes that normally contain the {gamma}{sub 3} and/or {alpha}{sub 5} subunits. The absence of an overt neurological phenotype in mice lacking the {gamma}{sub 3} and/or {alpha}{sub 5} subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.« less

Functional Expression of Two Neuronal Nicotinic Acetylcholine Receptors from cDNA Clones Identifies a Gene Family

NASA Astrophysics Data System (ADS)

Boulter, Jim; Connolly, John; Deneris, Evan; Goldman, Dan; Heinemann, Steven; Patrick, Jim

1987-11-01

A family of genes coding for proteins homologous to the α subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha3, alpha4, and beta2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha3 or alpha4 protein in combination with the beta2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by α -bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha3, alpha4, and beta2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha3, alpha4, and beta2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.

Studies on G-protein alpha.betagamma heterotrimer formation reveal a putative S-prenyl-binding site in the alpha subunit.

PubMed Central

Dietrich, Alexander; Scheer, Alexander; Illenberger, Daria; Kloog, Yoel; Henis, Yoav I; Gierschik, Peter

2003-01-01

The alpha and betagamma subunits of heterotrimeric G-proteins contain specific lipid modifications, which are required for their biological function. However, the relevance of these modifications to the interactions within the heterotrimeric G-protein is not fully understood. In order to explore the role of the S-prenyl moiety of the isoprenylated betagamma dimer of retinal transducin, betagamma(t), in the formation of the heterotrimeric complex with the corresponding N-acylated alpha subunit, alpha(t), we employed purified fully processed subunits, which are soluble in aqueous solutions without detergents. Pertussis-toxin-mediated [(32)P]ADP-ribosylation of alpha(t) is strongly stimulated (approximately 10-fold) in the presence of betagamma(t) and can thus serve as a measure for heterotrimer formation. Using this assay, preincubation of alpha(t) with S-prenyl analogues containing farnesyl or geranylgeranyl moieties was found to inhibit heterotrimer formation in a dose-dependent manner. The inhibition was competitive and reversible, as indicated by its reversal upon increase of the betagamma(t) dimer concentration or by removal of the S-prenyl analogue using gel filtration. The competitive nature of the inhibition is supported by the marked attenuation of the inhibition when the S-prenyl analogue was added to alpha(t) together with or after betagamma(t). The inhibition does not involve interaction with the alpha(t) acyl group, since an S-prenyl analogue inhibited the [(32)P]ADP-ribosylation of an unlipidated alpha(t) mutant. These data suggest the existence of a hitherto unrecognized S-prenyl-binding site in alpha(t), which is critical for its interaction with prenylated betagamma(t). PMID:12952523

Changes in proteasome structure and function caused by HAMLET in tumor cells.

PubMed

Gustafsson, Lotta; Aits, Sonja; Onnerfjord, Patrik; Trulsson, Maria; Storm, Petter; Svanborg, Catharina

2009-01-01

Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death. HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (beta1 and beta5) and structural subunits (alpha2, alpha3, alpha6 and beta3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells. The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.

Asymmetric processing of a substrate protein in sequential allosteric cycles of AAA+ nanomachines

NASA Astrophysics Data System (ADS)

Kravats, Andrea N.; Tonddast-Navaei, Sam; Bucher, Ryan J.; Stan, George

2013-09-01

Essential protein quality control includes mechanisms of substrate protein (SP) unfolding and translocation performed by powerful ring-shaped AAA+ (ATPases associated with various cellular activities) nanomachines. These SP remodeling actions are effected by mechanical forces imparted by AAA+ loops that protrude into the central channel. Sequential intra-ring allosteric motions, which underlie repetitive SP-loop interactions, have been proposed to comprise clockwise (CW), counterclockwise (CCW), or random (R) conformational transitions of individual AAA+ subunits. To probe the effect of these allosteric mechanisms on unfoldase and translocase functions, we perform Langevin dynamics simulations of a coarse-grained model of an all-alpha SP processed by the single-ring ClpY ATPase or by the double-ring p97 ATPase. We find that, in all three allosteric mechanisms, the SP undergoes conformational transitions along a common set of pathways, which reveals that the active work provided by the ClpY machine involves single loop-SP interactions. Nevertheless, the rates and yields of SP unfolding and translocation are controlled by mechanism-dependent loop-SP binding events, as illustrated by faster timescales of SP processing in CW allostery compared with CCW and R allostery. The distinct efficacy of allosteric mechanisms is due to the asymmetric collaboration of adjacent subunits, which involves CW-biased structural motions of AAA+ loops and results in CW-compatible torque applied onto the SP. Additional simulations of mutant ClpY rings, which render a subset of subunits catalytically-defective or reduce their SP binding affinity, reveal that subunit-based conformational transitions play the major role in SP remodeling. Based on these results we predict that the minimally functional AAA+ ring includes three active subunits, only two of which are adjacent.

Structural integration in hypoxia-inducible factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Dalei; Potluri, Nalini; Lu, Jingping

The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-alpha and ARNT (also called HIF-1 beta) subunits. Here we describe crystal structures for each of mouse HIF-2 alpha-ARNT and HIF-1 alpha-ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2 alpha-ARNT and HIF-1 alpha-ARNT, wherein ARNT spirals around the outside of each HIF-alpha subunit. Five distinctmore » pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-alpha mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.« less

The carboxyl-terminal region is a determinant for the intracellular behavior of the chorionic gonadotropin beta subunit: effects on the processing of the Asn-linked oligosaccharides.

PubMed

Muyan, M; Boime, I

1998-05-01

The placental hormone human CG (hCG) consists of two noncovalently linked alpha- and beta-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common alpha subunit but differ in their structurally distinct beta subunits. The CGbeta subunit is distinguished among the beta subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (carboxyl terminal peptide or CTP). In previous studies we observed that deleting this sequence decreased assembly of the truncated CGbeta subunit (CGbeta114) with the alpha-subunit and increased the heterogeneity of the secreted forms of the uncombined subunit synthesized in transfected Chinese hamster ovary (CHO) cells. The latter result was attributed to alterations in the processing of the two N-linked oligosaccharides. To examine at what step this heterogeneity occurs, the CGbeta and CGbeta114 genes were transfected into wild-type and mutant CHO cell lines that are defective in the late steps of the N-linked carbohydrate-processing pathway. We show here that removal of the CTP alters the processing of the core mannosyl unit of the subunit to complex forms at both glycosylation sites and that the oligosaccharides contain polylactosamine. Although it has been presumed that there is little intramolecular interaction between the CTP and the proximal domains of the subunit, our data suggest that the CTP sequence participates in the folding of the newly synthesized subunit, which is manifest by the posttranslational changes observed here.

Vacuolar H[sup +]-ATPase 69-kilodalton catalytic subunit cDNA from developing cotton (Gossypium hirsutum) ovules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilkins, T.A.

1993-06-01

This study investigates the molecular events of vacuole ontogeny in rapidly elongated cotton plant cells. Within the DNA coding region, the cotton and carrot cDNA clones exhibit 82.2% nucleotide sequence homology; at the amino acid level cotton and carrot catalytic subunits exhibited 95.7% identity and 2.1% amino acid similarity. When aligned with the analogous sequences from yeast, the cotton protein shared only 60.5% amino acid identity and 12.7% similarity. 10 refs., 1 tab.

A simple electrostatic switch important in the activation of type I protein kinase A by cyclic AMP.

PubMed

Vigil, Dominico; Lin, Jung-Hsin; Sotriffer, Christoph A; Pennypacker, Juniper K; McCammon, J Andrew; Taylor, Susan S

2006-01-01

Cyclic AMP activates protein kinase A by binding to an inhibitory regulatory (R) subunit and releasing inhibition of the catalytic (C) subunit. Even though crystal structures of regulatory and catalytic subunits have been solved, the precise molecular mechanism by which cyclic AMP activates the kinase remains unknown. The dynamic properties of the cAMP binding domain in the absence of cAMP or C-subunit are also unknown. Here we report molecular-dynamics simulations and mutational studies of the RIalpha R-subunit that identify the C-helix as a highly dynamic switch which relays cAMP binding to the helical C-subunit binding regions. Furthermore, we identify an important salt bridge which links cAMP binding directly to the C-helix that is necessary for normal activation. Additional mutations show that a hydrophobic "hinge" region is not as critical for the cross-talk in PKA as it is in the homologous EPAC protein, illustrating how cAMP can control diverse functions using the evolutionarily conserved cAMP-binding domains.

Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding.

PubMed

Churn, Severn B; Rana, Aniruddha; Lee, Kangmin; Parsons, J Travis; De Blas, Angel; Delorenzo, Robert J

2002-09-01

gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.

Methylation-regulated decommissioning of multimeric PP2A complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Cheng-Guo; Zheng, Aiping; Jiang, Li

2017-12-01

Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling pathway regulator) makes highly integrative multibranching contacts with the PP2A catalytic subunit, selective for the unmethylated tail and perturbing/inactivating the phosphatase active site. TIPRL also makes unusual wobble contacts with the scaffold subunit, allowing TIPRL, but not the overlapping regulatory subunits, to tolerate disease-associated PP2A mutations, resulting in reduced holoenzyme assembly and enhanced inactivationmore » of mutant PP2A. Strikingly, TIPRL and the latency chaperone, α4, coordinate to disassemble active holoenzymes into latent PP2A, strictly controlled by methylation. Our study reveals a mechanism for methylation-responsive inactivation and holoenzyme disassembly, illustrating the complexity of regulation/signaling, dynamic complex disassembly, and disease mutations in cancer and intellectual disability.« less

Phosphatidyl inositol 3-kinase signaling in hypothalamic proopiomelanocortin neurons contributes to the regulation of glucose homeostasis.

PubMed

Hill, Jennifer W; Xu, Yong; Preitner, Frederic; Fukuda, Makota; Cho, You-Ree; Luo, Ji; Balthasar, Nina; Coppari, Roberto; Cantley, Lewis C; Kahn, Barbara B; Zhao, Jean J; Elmquist, Joel K

2009-11-01

Recent studies demonstrated a role for hypothalamic insulin and leptin action in the regulation of glucose homeostasis. This regulation involves proopiomelanocortin (POMC) neurons because suppression of phosphatidyl inositol 3-kinase (PI3K) signaling in these neurons blunts the acute effects of insulin and leptin on POMC neuronal activity. In the current study, we investigated whether disruption of PI3K signaling in POMC neurons alters normal glucose homeostasis using mouse models designed to both increase and decrease PI3K-mediated signaling in these neurons. We found that deleting p85alpha alone induced resistance to diet-induced obesity. In contrast, deletion of the p110alpha catalytic subunit of PI3K led to increased weight gain and adipose tissue along with reduced energy expenditure. Independent of these effects, increased PI3K activity in POMC neurons improved insulin sensitivity, whereas decreased PI3K signaling resulted in impaired glucose regulation. These studies show that activity of the PI3K pathway in POMC neurons is involved in not only normal energy regulation but also glucose homeostasis.

Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs.

PubMed

Alvisi, Gualtiero; Ripalti, Alessandro; Ngankeu, Apollinaire; Giannandrea, Maila; Caraffi, Stefano G; Dias, Manisha M; Jans, David A

2006-10-01

The catalytic subunit of human cytomegalovirus (HCMV) DNA polymerase pUL54 is a 1242-amino-acid protein, whose function, stimulated by the processivity factor, phosphoprotein UL44 (ppUL44), is essential for viral replication. The C-terminal residues (amino acids 1220-1242) of pUL54 have been reported to be sufficient for ppUL44 binding in vitro. Although believed to be important for functioning in the nuclei of infected cells, no data are available on either the interaction of pUL54 with ppUL44 in living mammalian cells or the mechanism of pUL54 nuclear transport and its relationship with that of ppUL44. The present study examines for the first time the nuclear import pathway of pUL54 and its interaction with ppUL44 using dual color, quantitative confocal laser scanning microscopy on live transfected cells and quantitative gel mobility shift assays. We showed that of two nuclear localization signals (NLSs) located at amino acids 1153-1159 (NLSA) and 1222-1227 (NLSB), NLSA is sufficient to confer nuclear localization on green fluorescent protein (GFP) by mediating interaction with importin alpha/beta. We also showed that pUL54 residues 1213-1242 are sufficient to confer ppUL44 binding abilities on GFP and that pUL54 and ppUL44 can be transported to the nucleus as a complex. Our work thus identified distinct sites within the HCMV DNA polymerase, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import.

Ada protein-RNA polymerase sigma subunit interaction and alpha subunit-promoter DNA interaction are necessary at different steps in transcription initiation at the Escherichia coli Ada and aidB promoters.

PubMed

Landini, P; Bown, J A; Volkert, M R; Busby, S J

1998-05-22

The methylated form of the Ada protein (meAda) binds the ada and aidB promoters between 60 and 40 base pairs upstream from the transcription start and activates transcription of the Escherichia coli ada and aidB genes. This region is also a binding site for the alpha subunit of RNA polymerase and resembles the rrnB P1 UP element in A/T content and location relative to the core promoter. In this report, we show that deletion of the C-terminal domain of the alpha subunit severely decreases meAda-independent binding of RNA polymerase to ada and aidB, affecting transcription initiation at these promoters. We provide evidence that meAda activates transcription by direct interaction with the C-terminal domain of RNA polymerase sigma70 subunit (amino acids 574-613). Several negatively charged residues in the sigma70 C-terminal domain are important for transcription activation by meAda; in particular, a glutamic acid to valine substitution at position 575 has a dramatic effect on meAda-dependent transcription. Based on these observations, we propose that the role of the alpha subunit at ada and aidB is to allow initial binding of RNA polymerase to the promoters. However, transcription initiation is dependent on meAda-sigma70 interaction.

Control of the synthesis and subcellular targeting of the two GDH genes products in leaves and stems of Nicotiana plumbaginifolia and Arabidopsis thaliana.

PubMed

Fontaine, Jean-Xavier; Saladino, Francesca; Agrimonti, Caterina; Bedu, Magali; Tercé-Laforgue, Thérèse; Tétu, Thierry; Hirel, Bertrand; Restivo, Francesco M; Dubois, Frédéric

2006-03-01

Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an alpha- or a beta-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the alpha- and beta-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the alpha-subunit in the stems when the expression of the beta-subunit is impaired in the leaves, and of the beta-subunit in the leaves when the expression of the alpha-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.

Improve the catalytic activity of {alpha}-Fe{sub 2}O{sub 3} particles in decomposition of ammonium perchlorate by coating amorphous carbon on their surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Yifu; Liu Xinghai, E-mail: liuxh@whu.edu.c; Nie Jiaorong

2011-02-15

Sphere- and pod-like {alpha}-Fe{sub 2}O{sub 3} particles have been selectively synthesized using NH{sub 3}.H{sub 2}O and NaOH solution to adjust the pH value of the designed synthetic system, respectively. The sphere-like {alpha}-Fe{sub 2}O{sub 3} particles with diameter about 25 nm on average were encapsulated into carbon shells to fabricate a novel core-shell composite ({alpha}-Fe{sub 2}O{sub 3}-C) through the coating experiments. The catalytic performance of the products on the thermal decomposition of ammonium perchlorate (AP) was investigated by thermal gravimetric analyzer (TG) and differential thermal analysis (DTA). The thermal decomposition temperatures of AP in the presence of pod-like {alpha}-Fe{sub 2}O{sub 3},more » sphere-like {alpha}-Fe{sub 2}O{sub 3} and {alpha}-Fe{sub 2}O{sub 3}-C are reduced by 72, 81 and 109 {sup o}C, respectively, which show that {alpha}-Fe{sub 2}O{sub 3}-C core-shell composites have higher catalytic activity than that of {alpha}-Fe{sub 2}O{sub 3}. -- Graphical abstract: The catalytic performance of pod-like {alpha}-Fe{sub 2}O{sub 3}, sphere-like {alpha}-Fe{sub 2}O{sub 3} and {alpha}-Fe{sub 2}O{sub 3}-C on the thermal decomposition of ammonium perchlorate (AP). Display Omitted Research highlights: {yields} Sphere- and pod-like {alpha}-Fe{sub 2}O{sub 3} particles have been selectively synthesized using NH{sub 3}.H{sub 2}O and NaOH solution to adjust the pH value. {yields} A novel core-shell composite ({alpha}-Fe{sub 2}O{sub 3}-C core-shell structured composite) has been successfully synthesized using sphere-like {alpha}-Fe{sub 2}O{sub 3} particles as the cores and glucose as the source of carbon. {yields} The thermal decomposition temperatures of AP in the presence of pod-like {alpha}-Fe{sub 2}O{sub 3}, sphere-like {alpha}-Fe{sub 2}O{sub 3} and {alpha}-Fe{sub 2}O{sub 3}-C are reduced by 72, 81 and 109 {sup o}C, respectively, which shows that these materials have high catalytic activity.« less

Thylakoid membrane protein topography: transmembrane orientation of the chloroplast cytochrome b-559 psbE gene product.

PubMed

Tae, G S; Black, M T; Cramer, W A; Vallon, O; Bogorad, L

1988-12-27

Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the Mr 10,000 psbE gene product (alpha subunit) of the chloroplast cytochrome b-559. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the alpha subunit to a slightly smaller polypeptide (delta Mr approximately -1000) as detected on Western blots, without loss of reactivity to COOH-terminal antibody. The disappearance of the parent Mr 10,000 polypeptide from thylakoids in the presence of trypsin correlated with the appearance of the smaller polypeptide with delta Mr = -750, the conversion having a half-time of approximately 15 min. Exposure of inside-out vesicles to trypsin resulted in almost complete loss of reactivity to the antibody, showing that the COOH terminus is exposed on the lumenal side of the membrane. Removal of the extrinsic polypeptides of the oxygen-evolving complex resulted in an increase of the accessibility of the alpha subunit to trypsin. These data establish that the alpha subunit of cytochrome b-559 crosses the membrane once, as predicted from its single, 26-residue, hydrophobic domain. The NH2 terminus of the alpha polypeptide is on the stromal side of the membrane, where it is accessible, most likely at Arg-7 or Glu-6/Asp-11, to trypsin or V8 protease, respectively. As a consequence of this orientation, the single histidine residue in the alpha subunit is located on the stromal side of the hydrophobic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis.

PubMed

Aoubala, M; Holt, J; Clegg, R A; Rowlands, D J; Harris, M

2001-07-01

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

Fluorescence Polarization and Fluctuation Analysis Monitors Subunit Proximity, Stoichiometry, and Protein Complex Hydrodynamics

PubMed Central

Nguyen, Tuan A.; Sarkar, Pabak; Veetil, Jithesh V.; Koushik, Srinagesh V.; Vogel, Steven S.

2012-01-01

Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1–10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9±1.2 subunit, but values ranged from 10–14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2±1.3 subunits in the holoenzyme with values ranging from 9–12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved. PMID:22666486

Molecular basis of maple syrup urine disease: Novel mutations at the E1[alpha] locus that impair E1([alpha][sub 2][beta][sub 2]) assembly or decrease steady-state E1[alpha] mRNA levels of branched-chain [alpha]-keto acid dehydrogenase complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, J.L.; Fisher, C.R.; Chuang, D.T.

1994-08-01

The authors report the occurrence of three novel mutations in the E1[alpha] (BCKDHA) locus of the branched-chain [alpha]-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1[alpha] gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1[alpha] subunit. Both the 8-bp deletion and the single C insertionmore » generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1[alpha] mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1[alpha] subunit impairs its proper assembly with the normal E1[beta]. Unassembled as well as misassembled E1[alpha] and E1[beta] subunits are degraded in the cell. 32 refs., 8 figs.« less

Transgenic Over Expression of Nicotinic Receptor Alpha 5, Alpha 3, and Beta 4 Subunit Genes Reduces Ethanol Intake in Mice

PubMed Central

Gallego, Xavier; Ruiz, Jessica; Valverde, Olga; Molas, Susanna; Robles, Noemí; Sabrià, Josefa; Crabbe, John C.; Dierssen, Mara

2012-01-01

Abuse of alcohol and smoking are extensively co-morbid. Some studies suggest partial commonality of action of alcohol and nicotine mediated through nicotinic acetylcholine receptors (nAChRs). We tested mice with transgenic over expression of the alpha 5, alpha 3, beta 4 receptor subunit genes, which lie in a cluster on human chromosome 15, that were previously shown to have increased nicotine self-administration, for several responses to ethanol. Transgenic and wild-type mice did not differ in sensitivity to several acute behavioral responses to ethanol. However, transgenic mice drank less ethanol than wild-type in a two-bottle (ethanol vs. water) preference test. These results suggest a complex role for this receptor subunit gene cluster in the modulation of ethanol’s as well as nicotine’s effects. PMID:22459873

Deregulation of DNA-dependent protein kinase catalytic subunit contributes to human hepatocarcinogenesis development and has a putative prognostic value.

PubMed

Evert, M; Frau, M; Tomasi, M L; Latte, G; Simile, M M; Seddaiu, M A; Zimmermann, A; Ladu, S; Staniscia, T; Brozzetti, S; Solinas, G; Dombrowski, F; Feo, F; Pascale, R M; Calvisi, D F

2013-11-12

The DNA-repair gene DNA-dependent kinase catalytic subunit (DNA-PKcs) favours or inhibits carcinogenesis, depending on the cancer type. Its role in human hepatocellular carcinoma (HCC) is unknown. DNA-dependent protein kinase catalytic subunit, H2A histone family member X (H2AFX) and heat shock transcription factor-1 (HSF1) levels were assessed by immunohistochemistry and/or immunoblotting and qRT-PCR in a collection of human HCC. Rates of proliferation, apoptosis, microvessel density and genomic instability were also determined. Heat shock factor-1 cDNA or DNA-PKcs-specific siRNA were used to explore the role of both genes in HCC. Activator protein 1 (AP-1) binding to DNA-PKcs promoter was evaluated by chromatin immunoprecipitation. Kaplan-Meier curves and multivariate Cox model were used to study the impact on clinical outcome. Total and phosphorylated DNA-PKcs and H2AFX were upregulated in HCC. Activated DNA-PKcs positively correlated with HCC proliferation, genomic instability and microvessel density, and negatively with apoptosis and patient's survival. Proliferation decline and massive apoptosis followed DNA-PKcs silencing in HCC cell lines. Total and phosphorylated HSF1 protein, mRNA and activity were upregulated in HCC. Mechanistically, we demonstrated that HSF1 induces DNA-PKcs upregulation through the activation of the MAPK/JNK/AP-1 axis. DNA-dependent protein kinase catalytic subunit transduces HSF1 effects in HCC cells, and might represent a novel target and prognostic factor in human HCC.

Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin.

PubMed

Rivera, R T; Pasion, S G; Wong, D T; Fei, Y B; Biswas, D K

1989-06-01

A clonal strain of human lung tumor cells in culture (ChaGo), derived from a bronchogenic carcinoma, synthesizes and secretes large amounts of alpha (alpha) and a comparatively lower level of beta (beta) subunit of the glycoprotein hormone, human chorionic gonadotropin (HCG). ChaGo cells lost their characteristic anchorage-independent growth phenotype in the presence of anti-alpha-HCG antibody. The effect of the antibody was partially reversed by addition of alpha-HCG to the culture medium. ChaGo cells were transfected with an expression vector (pRSV-anti-alpha-HCG), that directs synthesis of RNA complementary to alpha-HCG mRNA. The transfectants produced alpha-HCG antisense RNA which was associated with the reduced level of alpha-HCG. Transfectants also displayed several altered phenotypic properties, including altered morphology, less mitosis, reduced growth rate, loss of anchorage-independent growth, and loss of tumorigenicity in nude mice. Treatment of transfectants with 8,bromo-cAMP resulted in increased accumulation of alpha-HCG mRNA, no change in the level of alpha-HCG antisense RNA, release of the inhibition of [3H]thymidine incorporation, and restoration of anchorage-independent growth phenotype. The overexpression of c-myc, observed in ChaGo cells, was unaffected by the reduced level of alpha-HCG. These results suggest that ectopic synthesis of the alpha subunit of HCG plays a functional role in the transformation of these human lung cells.

Chimeras of the integrin beta subunit mid-region reveal regions required for heterodimer formation and for activation.

PubMed

Hyland, R H; Douglass, W A; Tan, S M; Law, S K

2001-01-01

A central region of the beta2 integrin subunit, RN (residues D300 to C459), was replaced by the equivalent sequences from beta1 and beta7 to give the chimeras beta2RN1 and beta2RN7. Whilst the former construct failed to form heterodimer at the cell surface with alphaL, the later of these could be expressed together with the alphaL subunit to form a variant LFA-1. Based on recent modelling work, the RN region consists of two parts, one is the C-terminal end of the putative A-domain (RB, residues D300 to A359), and the other the mid-region (BN, residues Y360 to C459). Chimeras exchanging the two component regions were made. Of the four resultant chimeras, only the beta2RB1 chimera failed to support LFA-1 expression. Thus the beta1 specific residues of this region affect the interaction with the alphaL subunit. Whereas the alphaL/beta2RB7 LFA-1 variant is wildtype like with respect to ICAM-1 adhesion, the alphaLbeta2BN1 and alphaLbeta2BN7, as well as the alphaLbeta2RN7, variants are more adhesive than the wildtype. These results suggest that an authentic beta2 mid-region is, in part, required for maintaining the LFA-1 in a resting state.

Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

PubMed

Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

1997-06-25

Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

Crystal structure of heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ida, Koh; E-mail: idakoh@sci.kitasato-u.ac.jp; Moriguchi, Tomotaka

2005-07-29

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme. Here we report the crystal structures of the enzyme in complex with dimethylglycine and folinic acid. The {alpha} subunit is composed of two domains, contains NAD{sup +}, and binds folinic acid. The {beta} subunit contains dimethylglycine, FAD, and FMN, and these flavins are approximately 10 A apart. The {gamma} subunit is in contact with two domains of {alpha} subunit and has possibly a folate-binding structure. The {delta} subunit contains a single atom of zinc and has a Cys{sub 3}His zinc finger structure. Based on the structures determined and on themore » previous works, the structure-function relationship on the heterotetrameric sarcosine oxidase is discussed.« less

Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

PubMed

Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya

2008-11-01

A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

The Val192Leu mutation in the alpha-subunit of beta-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease.

PubMed Central

Hou, Y.; Vavougios, G.; Hinek, A.; Wu, K. K.; Hechtman, P.; Kaplan, F.; Mahuran, D. J.

1996-01-01

Substitution mutations adversely affecting the alpha-subunit of beta-hexosaminidase A (alphabeta) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-alpha chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an "active-site" residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all alpha-specific activity. This biochemical phenotype is referred to as the "B1-variant form" of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, alpha-Val192Leu. Chinese hamster ovary cells were permanently cotransfected with an alpha-cDNA-construct encoding the substitution and a mutant beta-cDNA (beta-Arg211Lys), encoding a beta-subunit that is inactive but normal in all other respects. We were surprised to find that the Val192Leu substitution, produced a pro-alpha chain that did not form alpha-beta dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val192Leu substitution does not specifically affect the alpha-active site. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8659543

The Val192Leu mutation in the alpha-subunit of beta-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease.

PubMed

Hou, Y; Vavougios, G; Hinek, A; Wu, K K; Hechtman, P; Kaplan, F; Mahuran, D J

1996-07-01

Substitution mutations adversely affecting the alpha-subunit of beta-hexosaminidase A (alphabeta) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-alpha chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an "active-site" residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all alpha-specific activity. This biochemical phenotype is referred to as the "B1-variant form" of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, alpha-Val192Leu. Chinese hamster ovary cells were permanently cotransfected with an alpha-cDNA-construct encoding the substitution and a mutant beta-cDNA (beta-Arg211Lys), encoding a beta-subunit that is inactive but normal in all other respects. We were surprised to find that the Val192Leu substitution, produced a pro-alpha chain that did not form alpha-beta dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val192Leu substitution does not specifically affect the alpha-active site.

Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the alpha6-integrin subunit.

PubMed

Kashimata, M; Gresik, E W

1997-02-01

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate branching morphogenesis of fetal mouse submandibular gland (SMG) rudiments in vitro. The EGF system (EGF, TGF-alpha, and their shared receptor, EGFR) also regulates expression of integrins and their ligands in the extracellular matrix. We show here that inhibition of EGFR tyrosine-kinase activity by a tyrphostin retards in vitro development of SMGs. Using total RNA isolated from pooled SMGs taken from intact mouse fetuses, mRNA transcripts for EGF, TGF-alpha, and EGFR were detected by reverse transcription-polymerase chain reaction (RT-PCR), and age-dependent variations in the levels of these mRNA were quantitatively determined by nuclease protection assays. These findings suggest that the EGF system is operative in the in vivo development of this gland. alpha6-Integrin subunit was localized by immunofluorescence at the basal surface of epithelial cells. Branching morphogenesis of cultured SMG rudiments was inhibited by anti-alpha6 antibodies. Synthesis of alpha6-subunit in cultured SMGs, detected by metabolic labeling and immunoprecipitation, was increased by EGF and drastically reduced by tyrphostin. RT-PCR revealed that mRNAs for alpha6- and beta1- and beta4-integrin subunits are expressed at all ages between embryonic day 13 and postnatal day 7. These findings suggest that 1) the EGF system is a physiologic regulator of development of fetal mouse SMG, and 2) one mechanism by which it acts may be by regulating expression of integrins, which in turn control interaction of epithelial cells with the extracellular matrix.

De novo missense mutations in the NAA10 gene cause severe non-syndromic developmental delay in males and females

PubMed Central

Popp, Bernt; Støve, Svein I; Endele, Sabine; Myklebust, Line M; Hoyer, Juliane; Sticht, Heinrich; Azzarello-Burri, Silvia; Rauch, Anita; Arnesen, Thomas; Reis, André

2015-01-01

Recent studies revealed the power of whole-exome sequencing to identify mutations in sporadic cases with non-syndromic intellectual disability. We now identified de novo missense variants in NAA10 in two unrelated individuals, a boy and a girl, with severe global developmental delay but without any major dysmorphism by trio whole-exome sequencing. Both de novo variants were predicted to be deleterious, and we excluded other variants in this gene. This X-linked gene encodes N-alpha-acetyltransferase 10, the catalytic subunit of the NatA complex involved in multiple cellular processes. A single hypomorphic missense variant p.(Ser37Pro) was previously associated with Ogden syndrome in eight affected males from two different families. This rare disorder is characterized by a highly recognizable phenotype, global developmental delay and results in death during infancy. In an attempt to explain the discrepant phenotype, we used in vitro N-terminal acetylation assays which suggested that the severity of the phenotype correlates with the remaining catalytic activity. The variant in the Ogden syndrome patients exhibited a lower activity than the one seen in the boy with intellectual disability, while the variant in the girl was the most severe exhibiting only residual activity in the acetylation assays used. We propose that N-terminal acetyltransferase deficiency is clinically heterogeneous with the overall catalytic activity determining the phenotypic severity. PMID:25099252

Role of phosphoinositide 3-kinase regulatory isoforms in development and actin rearrangement.

PubMed

Brachmann, Saskia M; Yballe, Claudine M; Innocenti, Metello; Deane, Jonathan A; Fruman, David A; Thomas, Sheila M; Cantley, Lewis C

2005-04-01

Class Ia phosphoinositide 3-kinases (PI3Ks) are heterodimers of p110 catalytic and p85 regulatory subunits that mediate a variety of cellular responses to growth and differentiation factors. Although embryonic development is not impaired in mice lacking all isoforms of the p85alpha gene (p85alpha-/- p55alpha-/- p50alpha-/-) or in mice lacking the p85beta gene (p85beta-/-) (D. A. Fruman, F. Mauvais-Jarvis, D. A. Pollard, C. M. Yballe, D. Brazil, R. T. Bronson, C. R. Kahn, and L. C. Cantley, Nat Genet. 26:379-382, 2000; K. Ueki, C. M. Yballe, S. M. Brachmann, D. Vicent, J. M. Watt, C. R. Kahn, and L. C. Cantley, Proc. Natl. Acad. Sci. USA 99:419-424, 2002), we show here that loss of both genes results in lethality at embryonic day 12.5 (E12.5). The phenotypes of these embryos, including subepidermal blebs flanking the neural tube at E8 and bleeding into the blebs during the turning process, are similar to defects observed in platelet-derived growth factor receptor alpha null (PDGFRalpha-/-) mice (P. Soriano, Development 124:2691-2700, 1997), suggesting that PI3K is an essential mediator of PDGFRalpha signaling at this developmental stage. p85alpha-/- p55alpha+/+ p50alpha+/+ p85beta-/- mice had similar but less severe defects, indicating that p85alpha and p85beta have a critical and redundant function in development. Mouse embryo fibroblasts deficient in all p85alpha and p85beta gene products (p85alpha-/- p55alpha-/- p50alpha-/- p85beta-/-) are defective in PDGF-induced membrane ruffling. Overexpression of the Rac-specific GDP-GTP exchange factor Vav2 or reintroduction of p85alpha or p85beta rescues the membrane ruffling defect. Surprisingly, reintroduction of p50alpha also restored PDGF-dependent membrane ruffling. These results indicate that class Ia PI3K is critical for PDGF-dependent actin rearrangement but that the SH3 domain and the Rho/Rac/Cdc42-interacting domain of p85, which lacks p50alpha, are not required for this response.

Secondary reduction of alpha7B integrin in laminin alpha2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle.

PubMed

Cohn, R D; Mayer, U; Saher, G; Herrmann, R; van der Flier, A; Sonnenberg, A; Sorokin, L; Voit, T

1999-03-01

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the beta1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of beta1D integrin was significantly reduced in the alpha7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of alpha7B in laminin alpha2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in alpha7 deficient mice, implies that the alpha7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

In vivo mechanism-based inactivation of S-adenosylmethionine decarboxylases from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae.

PubMed

Li, Y F; Hess, S; Pannell, L K; White Tabor, C; Tabor, H

2001-09-11

S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form alpha and beta subunits. The alpha subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity. With the use of an Escherichia coli overexpression system, we have purified AdoMetDCs encoded by the E. coli, Saccharomyces cerevisiae, and Salmonella typhimurium genes. Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally in vivo by a mechanism-based "suicide" inactivation. A large percentage of the alpha subunit of each enzyme had been modified in vivo to give peaks with masses m/z = 57 +/- 1 and m/z = 75 +/- 1 daltons higher than the parent peak. AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the alpha subunit. Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 +/- 1 adduct, which is probably derived from the reaction product. Comparable modification of the alpha subunit was also observed in in vitro experiments after incubation with the substrate or with the reaction product, which is consistent with the in vitro alkylation of E. coli AdoMetDC reported by Diaz and Anton [Diaz, E. & Anton, D. L. (1991) Biochemistry 30, 4078-4081].

Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis.

PubMed

Lemieux, M Joanne; Mark, Brian L; Cherney, Maia M; Withers, Stephen G; Mahuran, Don J; James, Michael N G

2006-06-16

Lysosomal beta-hexosaminidase A (Hex A) is essential for the degradation of GM2 gangliosides in the central and peripheral nervous system. Accumulation of GM2 leads to severely debilitating neurodegeneration associated with Tay-Sachs disease (TSD), Sandoff disease (SD) and AB variant. Here, we present the X-ray crystallographic structure of Hex A to 2.8 A resolution and the structure of Hex A in complex with NAG-thiazoline, (NGT) to 3.25 A resolution. NGT, a mechanism-based inhibitor, has been shown to act as a chemical chaperone that, to some extent, prevents misfolding of a Hex A mutant associated with adult onset Tay Sachs disease and, as a result, increases the residual activity of Hex A to a level above the critical threshold for disease. The crystal structure of Hex A reveals an alphabeta heterodimer, with each subunit having a functional active site. Only the alpha-subunit active site can hydrolyze GM2 gangliosides due to a flexible loop structure that is removed post-translationally from beta, and to the presence of alphaAsn423 and alphaArg424. The loop structure is involved in binding the GM2 activator protein, while alphaArg424 is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. The beta-subunit lacks these key residues and has betaAsp452 and betaLeu453 in their place; the beta-subunit therefore cleaves only neutral substrates efficiently. Mutations in the alpha-subunit, associated with TSD, and those in the beta-subunit, associated with SD are discussed. The effect of NGT binding in the active site of a mutant Hex A and its effect on protein function is discussed.

Molecular cloning and nucleotide sequence of the alpha and beta subunits of allophycocyanin from the cyanelle genome of Cyanophora paradoxa.

PubMed Central

Bryant, D A; de Lorimier, R; Lambert, D H; Dubbs, J M; Stirewalt, V L; Stevens, S E; Porter, R D; Tam, J; Jay, E

1985-01-01

The genes for the alpha- and beta-subunit apoproteins of allophycocyanin (AP) were isolated from the cyanelle genome of Cyanophora paradoxa and subjected to nucleotide sequence analysis. The AP beta-subunit apoprotein gene was localized to a 7.8-kilobase-pair Pst I restriction fragment from cyanelle DNA by hybridization with a tetradecameric oligonucleotide probe. Sequence analysis using that oligonucleotide and its complement as primers for the dideoxy chain-termination sequencing method confirmed the presence of both AP alpha- and beta-subunit genes on this restriction fragment. Additional oligonucleotide primers were synthesized as sequencing progressed and were used to determine rapidly the nucleotide sequence of a 1336-base-pair region of this cloned fragment. This strategy allowed the sequencing to be completed without a detailed restriction map and without extensive and time-consuming subcloning. The sequenced region contains two open reading frames whose deduced amino acid sequences are 81-85% homologous to cyanobacterial and red algal AP subunits whose amino acid sequences have been determined. The two open reading frames are in the same orientation and are separated by 39 base pairs. AP alpha is 5' to AP beta and both coding sequences are preceded by a polypurine, Shine-Dalgarno-type sequence. Sequences upstream from AP alpha closely resemble the Escherichia coli consensus promoter sequences and also show considerable homology to promoter sequences for several chloroplast-encoded psbA genes. A 56-base-pair palindromic sequence downstream from the AP beta gene could play a role in the termination of transcription or translation. The allophycocyanin apoprotein subunit genes are located on the large single-copy region of the cyanelle genome. PMID:2987916

Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.

PubMed Central

Shpakovski, G V; Acker, J; Wintzerith, M; Lacroix, J F; Thuriaux, P; Vigneron, M

1995-01-01

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit. PMID:7651387

The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma.

PubMed

Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J H; Okkenhaug, Klaus; Vanhaesebroeck, Bart

2008-06-17

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.

Low concentrations of ethanol do not affect radioligand binding to the delta-subunit-containing GABAA receptors in the rat brain.

PubMed

Mehta, Ashok K; Marutha Ravindran, C R; Ticku, Maharaj K

2007-08-24

In the present study, we investigated the co-localization pattern of the delta subunit with other subunits of GABA(A) receptors in the rat brain using immunoprecipitation and Western blotting techniques. Furthermore, we investigated whether low concentrations of ethanol affect the delta-subunit-containing GABA(A) receptor assemblies in the rat brain using radioligand binding to the rat brain membrane homogenates as well as to the immunoprecipitated receptor assemblies. Our results revealed that delta subunit is not co-localized with gamma(2) subunit but it is associated with the alpha(1), alpha(4) or alpha(6), beta(2) and/or beta(3) subunit(s) of GABA(A) receptors in the rat brain. Ethanol (1-50 mM) neither affected [(3)H]muscimol (3 nM) binding nor diazepam-insensitive [(3)H]Ro 15-4513 (2 nM) binding in the rat cerebellum and cerebral cortex membranes. However, a higher concentration of ethanol (500 mM) inhibited the binding of these radioligands to the GABA(A) receptors partially in the rat cerebellum and cerebral cortex. Similarly, ethanol (up to 50 mM) did not affect [(3)H]muscimol (15 nM) binding to the immunoprecipitated delta-subunit-containing GABA(A) receptor assemblies in the rat cerebellum and hippocampus but it inhibited the binding partially at a higher concentration (500 mM). These results suggest that the native delta-subunit-containing GABA(A) receptors do not play a major role in the pharmacology of clinically relevant low concentrations of ethanol.

Progesterone modulation of alpha5 nAChR subunits influences anxiety-related behavior during estrus cycle.

PubMed

Gangitano, D; Salas, R; Teng, Y; Perez, E; De Biasi, M

2009-06-01

Smokers often report an anxiolytic effect of cigarettes. In addition, stress-related disorders such as anxiety, post-traumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the alpha5 nicotinic acetylcholine receptor subunit in anxiety-related responses, control and alpha5 subunit null mice (alpha5(-/-)) were subjected to the open field activity (OFA), light-dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, alpha5(-/-) behaved like wild-type controls. In the EPM, female alpha5(-/-) mice displayed an anxiolytic-like phenotype, while male alpha5(-/-) mice were undistinguishable from littermate controls. We studied the hypothalamus-pituitary-adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin-releasing factor. Consistent with an anxiolytic-like phenotype, female alpha5(-/-) mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of alpha5, we treated cultured NTera 2 cells with progesterone and found that alpha5 protein levels were upregulated. In addition, brain levels of alpha5 mRNA increased upon progesterone injection into ovariectomized wild-type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolytic-like in wild-type mice, but no cycle-dependent fluctuations in anxiety levels were found in alpha5(-/-) females. Thus, alpha5-containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone-dependent modulation of alpha5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.

Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blanton, M.P.; Wang, H.H.

1990-02-06

A photoactivatable analogue of phosphatidylserine, {sup 125}I-labeled 4-azidosalicylic acid-phosphatidylserine ({sup 125}I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchRmore » {alpha} subunit that incorporated {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the {alpha} subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the {alpha} subunit incorporated little or no detectable amount of probe.« less

Glycosylation and processing of high-mannose oligosaccharides of thyroid-stimulating hormone subunits: comparison to nonsecretory cell glycoproteins.

PubMed

Ronin, C; Stannard, B S; Rosenbloom, I L; Magner, J A; Weintraub, B D

1984-09-25

Thyroid-stimulating hormone (TSH) subunit glycosylation was compared to that of total cell glycoproteins in mouse thyrotropic tumors. Lipid-linked oligosaccharides, total cell glycoproteins, and TSH subunits were labeled with either [3H]mannose, [3H]galactose, or [3H]glucose in pulse and pulse-chase experiments. The various oligosaccharides were isolated respectively by lipid extraction and mild acid hydrolysis, by selective immunoprecipitation, or by acid precipitation followed by trypsin and endoglycosidase H treatment. The nature of the oligosaccharides was assessed by their migration in paper chromatography, their relative incorporation of different precursors, and also their resistance to alpha-mannosidase. At 60 min, lipid-linked oligosaccharides were found to be composed of Glc3-2Man9GlcNAc2, Man9-8GlcNAc2, and Man5GlcNAc2. At 10 or 60 min of labeling, total cell proteins contained Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, Man9GlcNAc2, Glc1Man8GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2. The largest oligosaccharide, Glc3Man9GlcNAc2, had an unusually long half-life of about 2 h. In contrast, no Glc3Man9GlcNAc2 was found either on TSH + alpha subunits or on free beta subunits isolated either by immunoprecipitation or by sodium dodecyl sulfate gel electrophoresis. Instead, primarily Man9GlcNAc2 was found after a 10-min pulse both on TSH + alpha subunits and on beta subunits. When the pulse was followed by a chase up to 2 h, there was a progressive increase in Man8GlcNAc2 in higher amounts on TSH + alpha-subunit carbohydrate chains than on beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

PubMed

Wieczorek, Anna; McHenry, Charles S

2006-05-05

The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine, a novel photoaffinity antagonist.

PubMed

Chiara, David C; Trinidad, Jonathan C; Wang, Dong; Ziebell, Michael R; Sullivan, Deirdre; Cohen, Jonathan B

2003-01-21

[(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.

Integrin alphaIIb-subunit cytoplasmic domain mutations demonstrate a requirement for tyrosine phosphorylation of beta3-subunits in actin cytoskeletal organization.

PubMed

Yamodo, Innocent H; Blystone, Scott D

2004-01-01

Using truncated or mutated alphaIIb integrin cytoplasmic domains fused to the alphaV extracellular domain and expressed with the beta3 integrin subunit, we demonstrate that the double mutation of proline residues 998 and 999 to alanine (PP998/999AA), previously shown to disturb the C-terminal conformation of the alphaIIb integrin cytoplasmic domain, prevents tyrosine phosphorylation of beta3 integrin induced by Arg-Gly-Asp peptide ligation. This mutation also inhibits integrin mediated actin assembly and cell adhesion to vitronectin. In contrast, progressive truncation of the alphaIIb-subunit cytoplasmic domain did not reproduce these effects. Interestingly, the PP998/999AA mutations of alphaIIb did not affect beta3 tyrosine phosphorylation, cell adhesion, or actin polymerization induced by manganese. Exogenous addition of manganese was sufficient to rescue beta3 phosphorylation, cell adhesion, and actin assembly in cells expressing the PP998/999AA mutation when presented with a vitronectin substrate. Further, induction of the high affinity conformation of this mutant beta3 integrin by incubation with either Arg-Gly-Asp peptide or exogenous manganese was equivalent. These results suggest that the extracellular structure of beta3 integrins in the high affinity conformation is not directly related to the structure of the cytoplasmic face of the integrin. Moreover, the requirement for beta3 phosphorylation is demonstrated without mutation of the beta3 subunit. In support of our previous hypothesis of a role for beta3 phosphorylation in adhesion, these studies demonstrate a strong correlation between beta3 tyrosine phosphorylation and assembly of a cytoskeleton competent to support firm cell adhesion.

Transgenic over expression of nicotinic receptor alpha 5, alpha 3, and beta 4 subunit genes reduces ethanol intake in mice.

PubMed

Gallego, Xavier; Ruiz-Medina, Jessica; Valverde, Olga; Molas, Susanna; Robles, Noemí; Sabrià, Josefa; Crabbe, John C; Dierssen, Mara

2012-05-01

Abuse of alcohol and smoking are extensively co-morbid. Some studies suggest partial commonality of action of alcohol and nicotine mediated through nicotinic acetylcholine receptors (nAChRs). We tested mice with transgenic over expression of the alpha 5, alpha 3, beta 4 receptor subunit genes, which lie in a cluster on human chromosome 15, that were previously shown to have increased nicotine self-administration, for several responses to ethanol. Transgenic and wild-type mice did not differ in sensitivity to several acute behavioral responses to ethanol. However, transgenic mice drank less ethanol than wild-type in a two-bottle (ethanol vs. water) preference test. These results suggest a complex role for this receptor subunit gene cluster in the modulation of ethanol's as well as nicotine's effects. Copyright © 2012. Published by Elsevier Inc.

The chorionic gonadotropin alpha-subunit gene is on human chromosome 18 in JEG cells.

PubMed Central

Hardin, J W; Riser, M E; Trent, J M; Kohler, P O

1983-01-01

The gene for the alpha subunit of human chorionic gonadotropin (hCG) has been tentatively assigned to human chromosome 18. This localization was accomplished through the use of Southern blot analysis. A full-length cDNA probe for the hCG alpha subunit and DNA isolated from a series of somatic hybrids between mouse and human cells were utilized to make this assignment. In addition, in situ hybridization with normal human peripheral blood lymphocytes as a source of human chromosomes and with the same cDNA probe confirmed this result. The presence of human chromosome 18 was required for the detection of DNA fragments characteristic of the alpha-hCG gene. These results are consistent with our previous observation that human chromosomes 10 and 18 are required for the production of hCG in cultured cells. Images PMID:6578509

The over-expression of the β2 catalytic subunit of the proteasome decreases homologous recombination and impairs DNA double-strand break repair in human cells.

PubMed

Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

2011-01-01

By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

PubMed Central

Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

2011-01-01

By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p. PMID:21660142

Atrial natriuretic peptide regulates lipid mobilization and oxygen consumption in human adipocytes by activating AMPK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Souza, Sandra C.; Chau, Mary D.L.; Yang, Qing

2011-07-08

Highlights: {yields} Treatment of differentiated human adipocytes with atrial natriuretic peptide (ANP) increased lipolysis and oxygen consumption by activating AMP-activated protein kinase (AMPK). {yields} ANP stimulated lipid mobilization by selective activation of the alpha2 subunit of AMPK and increased energy utilization through activation of both the alpha1 and alpha2 subunits of AMPK. {yields} ANP enhanced adipocyte mitochondrial oxidative capacity as evidenced by induction of oxidative mitochondrial genes and increase in oxygen consumption. {yields} Exposure of human adipocytes to fatty acids and (TNF{alpha}) induced insulin resistance and decreased expression of mitochondrial genes which was restored to normal by ANP. -- Abstract:more » Atrial natriuretic peptide (ANP) has been shown to regulate lipid and carbohydrate metabolism providing a possible link between cardiovascular function and metabolism by mediating the switch from carbohydrate to lipid mobilization and oxidation. ANP exerts a potent lipolytic effect via cGMP-dependent protein kinase (cGK)-I mediated-stimulation of AMP-activated protein kinase (AMPK). Activation of the ANP/cGK signaling cascade also promotes muscle mitochondrial biogenesis and fat oxidation. Here we demonstrate that ANP regulates lipid metabolism and oxygen utilization in differentiated human adipocytes by activating the alpha2 subunit of AMPK. ANP treatment increased lipolysis by seven fold and oxygen consumption by two fold, both of which were attenuated by inhibition of AMPK activity. ANP-induced lipolysis was shown to be mediated by the alpha2 subunit of AMPK as introduction of dominant-negative alpha2 subunit of AMPK attenuated ANP effects on lipolysis. ANP-induced activation of AMPK enhanced mitochondrial oxidative capacity as evidenced by a two fold increase in oxygen consumption and induction of mitochondrial genes, including carnitine palmitoyltransferase 1A (CPT1a) by 1.4-fold, cytochrome C (CytC) by 1.3-fold, and peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) by 1.4-fold. Treatment of human adipocytes with fatty acids and tumor necrosis factor {alpha} (TNF{alpha}) induced insulin resistance and down-regulation of mitochondrial genes, which was restored by ANP treatment. These results show that ANP regulates lipid catabolism and enhances energy dissipation through AMPK activation in human adipocytes.« less

Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells.

PubMed

Yuan, X J; Wang, J; Juhaszova, M; Golovina, V A; Rubin, L J

1998-04-01

K(+)-channel activity-mediated alteration of the membrane potential and cytoplasmic free Ca2+ concentration ([Ca2+]cyt) is a pivotal mechanism in controlling pulmonary vasomotor tone. By using combined approaches of patch clamp, imaging fluorescent microscopy, and molecular biology, we examined the electrophysiological properties of K+ channels and the role of different K+ currents in regulating [Ca2+]cyt and explored the molecular identification of voltage-gated K+ (KV)- and Ca(2+)-activated K+ (KCa)-channel genes expressed in pulmonary arterial smooth muscle cells (PASMC). Two kinetically distinct KV currents [IK(V)], a rapidly inactivating (A-type) and a noninactivating delayed rectifier, as well as a slowly activated KCa current [IK(Ca)] were identified. IK(V) was reversibly inhibited by 4-aminopyridine (5 mM), whereas IK(Ca) was significantly inhibited by charybdotoxin (10-20 nM). K+ channels are composed of pore-forming alpha-subunits and auxiliary beta-subunits. Five KV-channel alpha-subunit genes from the Shaker subfamily (KV1.1, KV1.2, KV1.4, KV1.5, and KV1.6), a KV-channel alpha-subunit gene from the Shab subfamily (KV2.1), a KV-channel modulatory alpha-subunit (KV9.3), and a KCa-channel alpha-subunit gene (rSlo), as well as three KV-channel beta-subunit genes (KV beta 1.1, KV beta 2, and KV beta 3) are expressed in PASMC. The data suggest that 1) native K+ channels in PASMC are encoded by multiple genes; 2) the delayed rectifier IK(V) may be generated by the KV1.1, KV1.2, KV1.5, KV1.6, KV2.1, and/or KV2.1/KV9.3 channels; 3) the A-type IK(V) may be generated by the KV1.4 channel and/or the delayed rectifier KV channels (KV1 subfamily) associated with beta-subunits; and 4) the IK(Ca) may be generated by the rSlo gene product. The function of the KV channels plays an important role in the regulation of membrane potential and [Ca2+]cyt in PASMC.

Role of the HoxZ subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha immobilized on electrodes.

PubMed

Sezer, Murat; Frielingsdorf, Stefan; Millo, Diego; Heidary, Nina; Utesch, Tillman; Mroginski, Maria-Andrea; Friedrich, Bärbel; Hildebrandt, Peter; Zebger, Ingo; Weidinger, Inez M

2011-09-01

The role of the diheme cytochrome b (HoxZ) subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase (MBH) heterotrimer from Ralstonia eutropha H16 has been investigated. The MBH in its native heterotrimeric state was immobilized on electrodes and subjected to spectroscopic and electrochemical analysis. Surface enhanced resonance Raman spectroscopy was used to monitor the redox and coordination state of the HoxZ heme cofactors while concomitant protein film voltammetric measurements gave insights into the catalytic response of the enzyme on the electrode. The entire MBH heterotrimer as well as its isolated HoxZ subunit were immobilized on silver electrodes coated with self-assembled monolayers of ω-functionalized alkylthiols, displaying the preservation of the native heme pocket structure and an electrical communication between HoxZ and the electrode. For the immobilized MBH heterotrimer, catalytic reduction of the HoxZ heme cofactors was observed upon H(2) addition. The catalytic currents of MBH with and without the HoxZ subunit were measured and compared with the heterogeneous electron transfer rates of the isolated HoxZ. On the basis of the spectroscopic and electrochemical results, we conclude that the HoxZ subunit under these artificial conditions is not primarily involved in the electron transfer to the electrode but plays a crucial role in stabilizing the enzyme on the electrode. © 2011 American Chemical Society

Protein Phosphatase 2A Interacts with the Na+,K+-ATPase and Modulates Its Trafficking by Inhibition of Its Association with Arrestin

PubMed Central

Kimura, Toru; Han, WonSun; Pagel, Philipp; Nairn, Angus C.; Caplan, Michael J.

2011-01-01

Background The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Na+,K+-ATPase. The catalytic subunit of the Na+,K+-ATPase includes several functional domains that determine its enzymatic and trafficking properties. Methodology/Principal Findings Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A) catalytic C-subunit is a specific Na+,K+-ATPase interacting protein. PP-2A C-subunit interacted with the Na+,K+-ATPase, but not with the homologous sequences of the H+,K+-ATPase. We confirmed that the Na+,K+-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs) are important regulators of G-protein coupled receptor (GPCR) signaling, and they also regulate Na+,K+-ATPase trafficking through direct association. PP2A inhibits association between the Na+,K+-ATPase and arrestin, and diminishes the effect of arrestin on Na+,K+-ATPase trafficking. GRK phosphorylates the Na+,K+-ATPase and PP2A can at least partially reverse this phosphorylation. Conclusions/Significance Taken together, these data demonstrate that the sodium pump belongs to a growing list of ion transport proteins that are regulated through direct interactions with the catalytic subunit of a protein phosphatase. PMID:22242112

Somatic Host Cell Alterations in HPV Carcinogenesis

PubMed Central

Litwin, Tamara R.; Clarke, Megan A.; Dean, Michael; Wentzensen, Nicolas

2017-01-01

High-risk human papilloma virus (HPV) infections cause cancers in different organ sites, most commonly cervical and head and neck cancers. While carcinogenesis is initiated by two viral oncoproteins, E6 and E7, increasing evidence shows the importance of specific somatic events in host cells for malignant transformation. HPV-driven cancers share characteristic somatic changes, including apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-driven mutations and genomic instability leading to copy number variations and large chromosomal rearrangements. HPV-associated cancers have recurrent somatic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and phosphatase and tensin homolog (PTEN), human leukocyte antigen A and B (HLA-A and HLA-B)-A/B, and the transforming growth factor beta (TGFβ) pathway, and rarely have mutations in the tumor protein p53 (TP53) and RB transcriptional corepressor 1 (RB1) tumor suppressor genes. There are some variations by tumor site, such as NOTCH1 mutations which are primarily found in head and neck cancers. Understanding the somatic events following HPV infection and persistence can aid the development of early detection biomarkers, particularly when mutations in precancers are characterized. Somatic mutations may also influence prognosis and treatment decisions. PMID:28771191

Somatic Host Cell Alterations in HPV Carcinogenesis.

PubMed

Litwin, Tamara R; Clarke, Megan A; Dean, Michael; Wentzensen, Nicolas

2017-08-03

High-risk human papilloma virus (HPV) infections cause cancers in different organ sites, most commonly cervical and head and neck cancers. While carcinogenesis is initiated by two viral oncoproteins, E6 and E7, increasing evidence shows the importance of specific somatic events in host cells for malignant transformation. HPV-driven cancers share characteristic somatic changes, including apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-driven mutations and genomic instability leading to copy number variations and large chromosomal rearrangements. HPV-associated cancers have recurrent somatic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ( PIK3CA ) and phosphatase and tensin homolog ( PTEN ), human leukocyte antigen A and B ( HLA-A and HLA-B ) -A/B , and the transforming growth factor beta (TGFβ) pathway, and rarely have mutations in the tumor protein p53 ( TP53 ) and RB transcriptional corepressor 1 ( RB1 ) tumor suppressor genes. There are some variations by tumor site, such as NOTCH1 mutations which are primarily found in head and neck cancers. Understanding the somatic events following HPV infection and persistence can aid the development of early detection biomarkers, particularly when mutations in precancers are characterized. Somatic mutations may also influence prognosis and treatment decisions.

Uncovering the stoichiometry of Pyrococcus furiosus RNase P, a multi-subunit catalytic ribonucleoprotein complex, by surface-induced dissociation and ion mobility mass spectrometry.

PubMed

Ma, Xin; Lai, Lien B; Lai, Stella M; Tanimoto, Akiko; Foster, Mark P; Wysocki, Vicki H; Gopalan, Venkat

2014-10-20

We demonstrate that surface-induced dissociation (SID) coupled with ion mobility mass spectrometry (IM-MS) is a powerful tool for determining the stoichiometry of a multi-subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg(2+). We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5' maturation. Previous step-wise, Mg(2+)-dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM-MS in resolving conformational heterogeneity and yielding insights on RNP assembly. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular analysis and functional expression of the human type E neuronal Ca2+ channel alpha 1 subunit.

PubMed

Schneider, T; Wei, X; Olcese, R; Costantin, J L; Neely, A; Palade, P; Perez-Reyes, E; Qin, N; Zhou, J; Crawford, G D

1994-01-01

A human brain alpha 1 Ca2+ channel subunit was cloned and expressed in Xenopus laevis oocytes. The open reading frame, encoding 2,312 amino acids, has high homology to the marine ray doe-1, the rat E-type, and the rabbit brain BII alpha 1 subunits. The amino and carboxy termini of this human.E-type alpha 1 subunit (alpha 1E) are most similar to the rabbit BII-1 splice variant, the remainder being colinear with the BII alpha 1 with the exception of two insertions, one of 43 amino acids in the C-terminus and another of 7 amino acids, found also in the rat alpha 1E, between domains II and III. Two potential Ca2+ binding sites are predicted from its primary structure. The expression of inward Ba2+ currents reveals voltage-dependent activation and inactivation measured by the cut-open oocyte vaseline-gap technique, with kinetics that correspond to that of a high-voltage-activated neuronal Ca2+ channel, and pharmacologic properties that resemble those of some low-voltage-activated neuronal Ca2+ currents. The human alpha 1E currents are insensitive to omega-conotoxin-GVIA (1 microM), omega-agatoxin-IVA (200 nM), a synthetic funnel web spider toxin (FTX, 20 microM), and Bay-K8644 (0.5 microM); they are inhibited 20% by high concentrations of methoxyverapamil and diltiazem, 65% by 0.1% crude funnel web spider venom and 100% by Ni2+ (IC50 = 30 nM). Single-channel records show a complex activity pattern with several apparent conductance states, the largest having a conductance of 14 pS.

Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dwyer, B.P.

1991-04-23

The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digestsmore » of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.« less

A reduction in hippocampal GABAA receptor alpha5 subunits disrupts the memory for location of objects in mice.

PubMed

Prut, L; Prenosil, G; Willadt, S; Vogt, K; Fritschy, J-M; Crestani, F

2010-07-01

The memory for location of objects, which binds information about objects to discrete positions or spatial contexts of occurrence, is a form of episodic memory particularly sensitive to hippocampal damage. Its early decline is symptomatic for elderly dementia. Substances that selectively reduce alpha5-GABA(A) receptor function are currently developed as potential cognition enhancers for Alzheimer's syndrome and other dementia, consistent with genetic studies implicating these receptors that are highly expressed in hippocampus in learning performance. Here we explored the consequences of reduced GABA(A)alpha5-subunit contents, as occurring in alpha5(H105R) knock-in mice, on the memory for location of objects. This required the behavioral characterization of alpha5(H105R) and wild-type animals in various tasks examining learning and memory retrieval strategies for objects, locations, contexts and their combinations. In mutants, decreased amounts of alpha5-subunits and retained long-term potentiation in hippocampus were confirmed. They exhibited hyperactivity with conserved circadian rhythm in familiar actimeters, and normal exploration and emotional reactivity in novel places, allocentric spatial guidance, and motor pattern learning acquisition, inhibition and flexibility in T- and eight-arm mazes. Processing of object, position and context memories and object-guided response learning were spared. Genotype difference in object-in-place memory retrieval and in encoding and response learning strategies for object-location combinations manifested as a bias favoring object-based recognition and guidance strategies over spatial processing of objects in the mutants. These findings identify in alpha5(H105R) mice a behavioral-cognitive phenotype affecting basal locomotion and the memory for location of objects indicative of hippocampal dysfunction resulting from moderately decreased alpha5-subunit contents.

Polyester synthases: natural catalysts for plastics.

PubMed Central

Rehm, Bernd H A

2003-01-01

Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with a conserved cysteine residue as catalytic nucleophile. This review provides a survey of the known biochemical features of these unique enzymes and their proposed catalytic mechanism. PMID:12954080

Oligomeric properties of alpha-dendrotoxin-sensitive potassium ion channels purified from bovine brain.

PubMed

Parcej, D N; Scott, V E; Dolly, J O

1992-11-17

Neuronal acceptors for alpha-dendrotoxin (alpha-DTX) have recently been purified from mammalian brain and shown to consist of two classes of subunit, a larger (approximately 78,000 M(r)) protein (alpha) whose N-terminal sequence is identical to that of a cloned, alpha-DTX-sensitive K+ channel, and a novel M(r) 39,000 (beta) polypeptide of unknown function. However, little information is available regarding the oligomeric composition of these native molecules. By sedimentation analysis of alpha-DTX acceptors isolated from bovine cortex, two species have been identified. A minority of these oligomers contain only the larger protein, while the vast majority possess both subunits. Based on accurate determination of the molecular weights of these two forms it is proposed that alpha-DTX-sensitive K+ channels exist as alpha 4 beta 4 complexes because this combination gives the best fit to the experimental data.

PKA catalytic subunit compartmentation regulates contractile and hypertrophic responses to β-adrenergic signaling

PubMed Central

Yang, Jason H.; Polanowska-Grabowska, Renata K.; Smith, Jeffrey S.; Shields, Charles W.; Saucerman, Jeffrey J.

2014-01-01

β-adrenergic signaling is spatiotemporally heterogeneous in the cardiac myocyte, conferring exquisite control to sympathetic stimulation. Such heterogeneity drives the formation of protein kinase A (PKA) signaling microdomains, which regulate Ca2+ handling and contractility. Here, we test the hypothesis that the nucleus independently comprises a PKA signaling microdomain regulating myocyte hypertrophy. Spatially-targeted FRET reporters for PKA activity identified slower PKA activation and lower isoproterenol sensitivity in the nucleus (t50 = 10.60±0.68 min; EC50 = 89.00 nmol/L) than in the cytosol (t50 = 3.71±0.25 min; EC50 = 1.22 nmol/L). These differences were not explained by cAMP or AKAP-based compartmentation. A computational model of cytosolic and nuclear PKA activity was developed and predicted that differences in nuclear PKA dynamics and magnitude are regulated by slow PKA catalytic subunit diffusion, while differences in isoproterenol sensitivity are regulated by nuclear expression of protein kinase inhibitor (PKI). These were validated by FRET and immunofluorescence. The model also predicted differential phosphorylation of PKA substrates regulating cell contractility and hypertrophy. Ca2+ and cell hypertrophy measurements validated these predictions and identified higher isoproterenol sensitivity for contractile enhancements (EC50 = 1.84 nmol/L) over cell hypertrophy (EC50 = 85.88 nmol/L). Over-expression of spatially targeted PKA catalytic subunit to the cytosol or nucleus enhanced contractile and hypertrophic responses, respectively. We conclude that restricted PKA catalytic subunit diffusion is an important PKA compartmentation mechanism and the nucleus comprises a novel PKA signaling microdomain, insulating hypertrophic from contractile β-adrenergic signaling responses. PMID:24225179

Arabidopsis PPP family of serine/threonine protein phosphatases: many targets but few engines.

PubMed

Uhrig, R Glen; Labandera, Anne-Marie; Moorhead, Greg B

2013-09-01

The major plant serine/threonine protein phosphatases belong to the phosphoprotein phosphatase (PPP) family. Over the past few years the complement of Arabidopsis thaliana PPP family of catalytic subunits has been cataloged and many regulatory subunits identified. Specific roles for PPPs have been characterized, including roles in auxin and brassinosteroid signaling, in phototropism, in regulating the target of rapamycin pathway, and in cell stress responses. In this review, we provide a framework for understanding the PPP family by exploring the fundamental role of the phosphatase regulatory subunits that drive catalytic engine specificity. Although there are fewer plant protein phosphatases compared with their protein kinase partners, their function is now recognized to be as dynamic and as regulated as that of protein kinases. Copyright © 2013 Elsevier Ltd. All rights reserved.

Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors

PubMed Central

Farroni, Jeffrey S; McCool, Brian A

2004-01-01

Background Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. Results While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The β-amino acid taurine possessed 30–50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for β-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. Conclusions Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology. PMID:15301692

Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors.

PubMed

Farroni, Jeffrey S; McCool, Brian A

2004-08-09

Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The beta-amino acid taurine possessed 30-50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for beta-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology.

The Val{sup 192}Leu mutation in the {alpha}-subunit of {beta}-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Y.; Vavougios, G.; Hinek, A.

1996-07-01

Substitution mutations adversely affecting the {alpha}-subunit of {beta}-hexosaminidase A ({alpha}{beta}) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-{alpha} chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an {open_quotes}active-site{close_quotes} residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all {alpha}-specific activity. This biochemical phenotype is referred to as the {open_quotes}B1-variant form{close_quotes} of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and bothmore » subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, {alpha}-Val{sup 192}Leu. Chinese hamster ovary cells were permanently cotransfected with an {alpha}-cDNA-construct encoding the substitution and a mutant {beta}-cDNA ({beta}-Arg{sup 211}Lys), encoding a {beta}-subunit that is inactive but normal in all other respects. We were surprised to find that the Val{sup 192}Leu substitution produced a pro-{alpha} chain that did not form {alpha}-{beta} dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val{sup 192}Leu substitution does not specifically affect the {alpha}-active site. 23 refs., 4 figs., 2 tabs.« less

Immunohistochemical analyses of alpha1 and alpha3 Na+/K+-ATPase subunit expression in medulloblastomas.

PubMed

Suñol, Mariona; Cusi, Victoria; Cruz, Ofelia; Kiss, Robert; Lefranc, Florence

2011-03-01

The levels of expression of the α1 and α3 subunits of the Na(+)/K(+)-ATPase (the NaK sodium pump) in medulloblastomas are unclear. This study investigated the expression of the NaK subunits using immunohistochemical methods in 29 medulloblastomas including 23 classic, three large-cell/anaplastic and three nodular/desmoplastic medulloblastomas, as well as in three atypical teratoid/rhabdoid tumors (AT/RTs). There was overexpression of the α1 or α3 NaK subunits in more than half of the medulloblastomas and atypical AT/RTs, with about one-third of these tumours displaying overexpression of both subunits. These preliminary data suggest that targeting these subunits in AT/RTs and medulloblastomas that overexpress these proteins may lead to therapeutic benefit. These findings warrant confirmation in larger numbers of patients than those used in this study. Moreover, it should be determined whether inhibition of the α1/α3 NaK subunits can be integrated into the risk stratification schemes already in use for medulloblastoma patients.

Use of polyclonal and monoclonal antibodies to study hCG-receptor interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Milius, R.P.

1985-01-01

Although the glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) bind to different receptors, each contains an identical alpha subunit. Specificity is somehow endowed by theta subunits which are distinct for each hormone. Human choriogonadotropin (hCG) is a natural LH analog that contains a beta subunit nearly identical to that of LH. The roles of these subunits in the recognition and high affinity binding of hCG to receptor was examined. Polyclonal and monoclonal antibodies specific for the individual subunits of hCG were used to probe the hormone-receptor interaction. Conformation-specific and sequence-specific antibodies were examined for their abilities to bindmore » Triton X-100-solubilized /sup 125/I-hCG-receptor complex and to inhibit hormone binding to crude rat ovarian membranes containing receptor. Even though the immunoreactive sites are not located on the receptor binding surface of the beta subunit, most, but not all, of these polyclonal and monoclonal antibodies were able to inhibit /sup 125/I-hCG binding to receptor. Although the inhibition of binding may be due to steric interference due to the size of the antibody molecules, a two-step model for hCG binding to receptor is presented that also explains these results. In this model, the beta subunit initially binds with the receptor with a highly specific but low affinity interaction. This activates a site for the high affinity binding of the alpha subunit and stabilization of the complex. This is an attractive model as it may be applied to other glycoprotein hormones sharing an alpha subunit.« less

Characterization of rat leydig cell gonadotropin receptor structure by affinity cross-linking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Q.Y.; Hwang, J.; Menon, K.M.J.

1986-05-01

The gonadotropin receptor from rat leydig cell has been characterized with respect to binding kinetics and physiological regulation. The present study was intended to examine the structure of the receptor. Leydig cell suspension was prepared by either collagenase digestion or by mechanical disruption of the testis. The cells were incubated with /sup 125/I-hCG and the unreacted hCG was removed by centrifugation. The /sup 125/I-hCG was then covalently linked to the cell surface receptor using cleavable (dithiobis (succinimidyl propionate)) and non-cleavable (disuccinimidyl suberate) cross-linking reagents. The extracted cross-linked membrane proteins were resolved on SDS-polyacrylamide gels under reducing and non-reducing conditions andmore » subjected to autoradiographic analysis. Under non-reducing conditions, two labeled species with M/sub r/ = 87,000 and 120,000 were detected. However, only one labeled band was detected under reducing conditions with M/sub r/ = 64,000. The binding of /sup 125/I-hCG to the receptor was inhibited by hCG and LH, but not by a number of peptides and proteins. The data suggest that hCG receptor in leydig cell is an oligomeric complex consisting of four subunits, ..cap alpha cap alpha beta gamma... The ..beta.. and ..gamma.. subunits are each linked to an ..cap alpha.. subunit through disulfide linkage and the hormone binds to each ..cap alpha.. subunit. The two dimers formed (..cap alpha beta cap alpha gamma..) are associated by noncovalent interactions.« less

Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism.

PubMed

Shirakihara, Yasuo; Shiratori, Aya; Tanikawa, Hiromi; Nakasako, Masayoshi; Yoshida, Masasuke; Suzuki, Toshiharu

2015-08-01

F1-ATPase (F1) is the catalytic sector in F(o)F1-ATP synthase that is responsible for ATP production in living cells. In catalysis, its three catalytic β-subunits undergo nucleotide occupancy-dependent and concerted open-close conformational changes that are accompanied by rotation of the γ-subunit. Bacterial and chloroplast F1 are inhibited by their own ε-subunit. In the ε-inhibited Escherichia coli F1 structure, the ε-subunit stabilizes the overall conformation (half-closed, closed, open) of the β-subunits by inserting its C-terminal helix into the α3β3 cavity. The structure of ε-inhibited thermophilic F1 is similar to that of E. coli F1, showing a similar conformation of the ε-subunit, but the thermophilic ε-subunit stabilizes another unique overall conformation (open, closed, open) of the β-subunits. The ε-C-terminal helix 2 and hook are conserved between the two structures in interactions with target residues and in their positions. Rest of the ε-C-terminal domains are in quite different conformations and positions, and have different modes of interaction with targets. This region is thought to serve ε-inhibition differently. For inhibition, the ε-subunit contacts the second catches of some of the β- and α-subunits, the N- and C-terminal helices, and some of the Rossmann fold segments. Those contacts, as a whole, lead to positioning of those β- and α- second catches in ε-inhibition-specific positions, and prevent rotation of the γ-subunit. Some of the structural features are observed even in IF1 inhibition in mitochondrial F1. © 2015 FEBS.

Nuclear RNA Exosome at 3.1 Å Reveals Substrate Specificities, RNA Paths, and Allosteric Inhibition of Rrp44/Dis3.

PubMed

Zinder, John C; Wasmuth, Elizabeth V; Lima, Christopher D

2016-11-17

The eukaryotic RNA exosome is an essential and conserved 3'-to-5' exoribonuclease complex that degrades or processes nearly every class of cellular RNA. The nuclear RNA exosome includes a 9-subunit non-catalytic core that binds Rrp44 (Dis3) and Rrp6 subunits to modulate their processive and distributive 3'-to-5' exoribonuclease activities, respectively. Here we utilize an engineered RNA with two 3' ends to obtain a crystal structure of an 11-subunit nuclear exosome bound to RNA at 3.1 Å. The structure reveals an extended RNA path to Rrp6 that penetrates into the non-catalytic core; contacts between the non-catalytic core and Rrp44, which inhibit exoribonuclease activity; and features of the Rrp44 exoribonuclease site that support its ability to degrade 3' phosphate RNA substrates. Using reconstituted exosome complexes, we show that 3' phosphate RNA is not a substrate for Rrp6 but is readily degraded by Rrp44 in the nuclear exosome. Copyright © 2016 Elsevier Inc. All rights reserved.

Evolution and expression of the phosphodiesterase 6 genes unveils vertebrate novelty to control photosensitivity.

PubMed

Lagman, David; Franzén, Ilkin E; Eggert, Joel; Larhammar, Dan; Abalo, Xesús M

2016-06-13

Phosphodiesterase 6 (PDE6) is a protein complex that hydrolyses cGMP and acts as the effector of the vertebrate phototransduction cascade. The PDE6 holoenzyme consists of catalytic and inhibitory subunits belonging to two unrelated gene families. Rods and cones express distinct genes from both families: PDE6A and PDE6B code for the catalytic and PDE6G the inhibitory subunits in rods while PDE6C codes for the catalytic and PDE6H the inhibitory subunits in cones. We performed phylogenetic and comparative synteny analyses for both gene families in genomes from a broad range of animals. Furthermore, gene expression was investigated in zebrafish. We found that both gene families expanded from one to three members in the two rounds of genome doubling (2R) that occurred at the base of vertebrate evolution. The PDE6 inhibitory subunit gene family appears to be unique to vertebrates and expanded further after the teleost-specific genome doubling (3R). We also describe a new family member that originated in 2R and has been lost in amniotes, which we have named pde6i. Zebrafish has retained two additional copies of the PDE6 inhibitory subunit genes after 3R that are highly conserved, have high amino acid sequence identity, are coexpressed in the same photoreceptor type as their amniote orthologs and, interestingly, show strikingly different daily oscillation in gene expression levels. Together, these data suggest specialisation related to the adaptation to different light intensities during the day-night cycle, most likely maintaining the regulatory function of the PDE inhibitory subunits in the phototransduction cascade.

Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity.

PubMed

Bertenshaw, G P; Turk, B E; Hubbard, S J; Matters, G L; Bylander, J E; Crisman, J M; Cantley, L C; Bond, J S

2001-04-20

Meprin A and B are highly regulated, secreted, and cell-surface metalloendopeptidases that are abundantly expressed in the kidney and intestine. Meprin oligomers consist of evolutionarily related alpha and/or beta subunits. The work herein was carried out to identify bioactive peptides and proteins that are susceptible to hydrolysis by mouse meprins and kinetically characterize the hydrolysis. Gastrin-releasing peptide fragment 14-27 and gastrin 17, regulatory molecules of the gastrointestinal tract, were found to be the best peptide substrates for meprin A and B, respectively. Peptide libraries and a variety of naturally occurring peptides revealed that the meprin beta subunit has a clear preference for acidic amino acids in the P1 and P1' sites of substrates. The meprin alpha subunit selected for small (e.g. serine, alanine) or hydrophobic (e.g. phenylalanine) residues in the P1 and P1' sites, and proline was the most preferred amino acid at the P2' position. Thus, although the meprin alpha and beta subunits share 55% amino acid identity within the protease domain and are normally localized at the same tissue cell surfaces, they have very different substrate and peptide bond specificities indicating different functions. Homology models of the mouse meprin alpha and beta protease domains, based on the astacin crystal structure, revealed active site differences that can account for the marked differences in substrate specificity of the two subunits.

Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone alpha subunit gene expression.

PubMed

Sato, Takanobu; Kitahara, Kousuke; Susa, Takao; Kato, Takako; Kato, Yukio

2006-10-01

Recently, we have reported that a Prophet of Pit-1 homeodomain factor, Prop-1, is a novel transcription factor for the porcine follicle-stimulating hormone beta subunit (FSHbeta) gene. This study subsequently aimed to examine the role of Prop-1 in the gene expression of two other porcine gonadotropin subunits, pituitary glycoprotein hormone alpha subunit (alphaGSU), and luteinizing hormone beta subunit (LHbeta). A series of deletion mutants of the porcine alphaGSU (up to -1059 bp) and LHbeta (up to -1277 bp) promoters were constructed in the reporter vector, fused with the secreted alkaline phosphatase gene (pSEAP2-Basic). Transient transfection studies using GH3 cells were carried out to estimate the activation of the porcine alphaGSU and LHbeta promoters by Prop-1, which was found to activate the alphaGSU promoter of -1059/+12 bp up to 11.7-fold but not the LHbeta promoter. Electrophoretic mobility shift assay and DNase I footprinting analysis revealed that Prop-1 binds to six positions, -1038/-1026, -942/-928, -495/-479, -338/-326, -153/-146, and -131/-124 bp, that comprise the A/T cluster. Oligonucleotides of six Prop-1 binding sites were directly connected to the minimum promoter of alphaGSU, fused in the pSEAP2-Basic vector, followed by transfecting GH3 cells to determine the cis-acting activity. Finally, we concluded that at least five Prop-1 binding sites are the cis-acting elements for alphaGSU gene expression. The present results revealed a notable feature of the proximal region, where three Prop-1-binding sites are close to and/or overlap the pituitary glycoprotein hormone basal element, GATA-binding element, and junctional regulatory element. To our knowledge, this is the first demonstration of the role of Prop-1 in the regulation of alphaGSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHbeta gene, confirm that Prop-1 modulates the synthesis of FSH at the transcriptional level. On the other hand, the defects of Prop-1 are known to cause dwarfism and combined pituitary hormone deficiency accompanying hypogonadism. Accordingly, the present observations provide a novel view to understand the hypogonadism caused by Prop-1 defects at the molecular level through the regulatory mechanism of alphaGSU and FSHbeta gene expressions.

5-Fluoroindole Resistance Identifies Tryptophan Synthase Beta Subunit Mutants in Arabidopsis Thaliana

PubMed Central

Barczak, A. J.; Zhao, J.; Pruitt, K. D.; Last, R. L.

1995-01-01

A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation. PMID:7635295

Molecular characterization of the alpha subunit of complement component C8 (GcC8alpha) in the nurse shark (Ginglymostoma cirratum).

PubMed

Aybar, Lydia; Shin, Dong-Ho; Smith, Sylvia L

2009-09-01

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of alpha, beta, and gamma subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8alpha and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8gamma-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4% identity with human and Xenopus C8alpha respectively. Southern blot analysis showed GcC8alpha exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8alpha orthologs and as a sister taxa to the Xenopus. 2009 Elsevier Ltd.

The ionic track in the F1-ATPase from the thermophilic Bacillus PS3.

PubMed

Bandyopadhyay, Sanjay; Allison, William S

2004-03-09

Only beta-beta cross-links form when the alpha(3)(betaE(395)C)(3)gammaK(36)C (MF(1) residue numbers) double mutant subcomplex of TF(1), the F(1)-ATPase from the thermophilic Bacillus PS3, is slowly inactivated with CuCl(2) in the presence or absence of MgATP. The same slow rate of inactivation and extent of beta-beta cross-linking occur upon treatment of the alpha(3)(betaE(395)C)(3)gamma single mutant subcomplex with CuCl(2) under the same conditions. In contrast, the alpha(3)(betaE(395)C)(3)gammaR(33)C and alpha(3)(betaE(395)C)(3)gammaR(75)C double mutant subcomplexes of TF(1) are rapidly inactivated by CuCl(2) under the same conditions that is accompanied by complete beta-gamma cross-linking. The ATPase activity of each mutant enzyme containing the betaE(395)C substitution is stimulated to a much greater extent by the nonionic detergent lauryldimethylamine oxide (LDAO) than wild-type enzyme, whereas the ATPase activities of the gammaR(33)C, gammaK(36)C, and gammaR(75)C single mutants are stimulated to about the same extent as wild-type enzyme by LDAO. This indicates that the E(395)C substitution in the (394)DELSEED(400) segment of beta subunits increases propensity of the enzyme to entrap inhibitory MgADP in a catalytic site during turnover. These results are discussed in perspective with (i) the ionic track predicted from molecular dynamics simulations to operate during energy-driven ATP synthesis by MF(1), the F(1)-ATPase from bovine heart mitochondria [Ma, J., Flynn, T. C., Cui, Q., Leslie, A. G. W., Walker, J. E., and Karplus, M. (2002) Structure 10, 921-931]; and (ii) the possibility that the betaE(395)C substitution might induce a global effect that alters affinity of noncatalytic sites for nucleotides or alters communication between noncatalytic sites and catalytic sites during ATP hydrolysis.

Structure of Glycerol Dehydratase Reactivase: A New Type of Molecular Chaperone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao, Der-Ing; Reiss, Lisa; Turner, Jr., Ivan

2010-03-08

The function of glycerol dehydratase (GDH) reactivase is to remove damaged coenzyme B{sub 12} from GDH that has suffered mechanism-based inactivation. The structure of GDH reactivase from Klebsiella pneumoniae was determined at 2.4 {angstrom} resolution by the single isomorphous replacement with anomalous signal (SIR/AS) method. Each tetramer contains two elongated 63 kDa {alpha} subunits and two globular 14 kDa {beta} subunits. The {alpha} subunit contains structural features resembling both GroEL and Hsp70 groups of chaperones, and it appears chaperone like in its interactions with ATP. The fold of the {beta} subunit resembles that of the {beta} subunit of glycerol dehydratase,more » except that it lacks some coenzyme B12 binding elements. A hypothesis for the reactivation mechanism of reactivase is proposed based on these structural features.« less

Integrative Approach for Computationally Inferring Interactions between the Alpha and Beta Subunits of the Calcium-Activated Potassium Channel (BK): a Docking Study

PubMed Central

González, Janneth; Gálvez, Angela; Morales, Ludis; Barreto, George E.; Capani, Francisco; Sierra, Omar; Torres, Yolima

2013-01-01

Three-dimensional models of the alpha- and beta-1 subunits of the calcium-activated potassium channel (BK) were predicted by threading modeling. A recursive approach comprising of sequence alignment and model building based on three templates was used to build these models, with the refinement of non-conserved regions carried out using threading techniques. The complex formed by the subunits was studied by means of docking techniques, using 3D models of the two subunits, and an approach based on rigid-body structures. Structural effects of the complex were analyzed with respect to hydrogen-bond interactions and binding-energy calculations. Potential interaction sites of the complex were determined by referencing a study of the difference accessible surface area (DASA) of the protein subunits in the complex. PMID:23492851

Biochemical analysis of human POLG2 variants associated with mitochondrial disease

PubMed Central

Young, Matthew J.; Longley, Matthew J.; Li, Fang-Yuan; Kasiviswanathan, Rajesh; Wong, Lee-Jun; Copeland, William C.

2011-01-01

Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, eight heterozygous mutations (six novel) in POLG2 were identified in one control and eight patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations, we biochemically characterized seven variants [c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)] that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic. PMID:21555342

Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Masure, H.R.; Donovan, M.G.; Storm, D.R.

1991-01-01

An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca{sup 2}{sup +} to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increasesmore » in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca{sup 2}{sup +} and this interaction may be important for its invasion into animal cells.« less

Two subunits of the 55 K porcine zona pellucida glycoprotein family are immunologically distinct

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subramanian, M.G.; Yurewicz, E.C.; Sacco, A.G.

1986-03-01

The 55K glycoprotein family (ZP3) of the porcine zona pellucida is comprised of two subunits of 46 K and 45 K which can be resolved by endo-..beta..-galactosidase digestion of ZP3 followed by reversed phase HPLC on Vydac C4 resin. Gel electrophoresis revealed that the 46 K component (EBDG..cap alpha..) is approx. 95% pure and the 45 K component (EBGD..beta..) is 100% pure. In the present study, these two subunits were evaluated immunologically by RIA. Under similar reaction protocols (chloramine-T iodination procedure) comparable specific activities were obtained for EBGD..cap alpha.. (33.06 +/- 7.5 ..mu..ci/..mu..gm), EBGD..beta.. (30.45 +/- 1.6) and ZP3 (26.3more » +/- 1.3). Antibody (Ab) titration studies revealed that EBGD..cap alpha.. and ..beta.. are potent immunogens and /sup 125/I-EBGD..cap alpha.. showed minimal cross reactivity to EBGD..beta..-Ab (8% bound at 1:500 dilution), whereas, /sup 125/I-EBGD..beta.. showed a greater degree of cross reactivity to EBGD..cap alpha..-Ab (23% bound at 1:500 dilution). Maximum binding for the two labeled antigens against homologous Abs (1:500) was > 60%. Dose response studies revealed that in the /sup 125/I-EBGD..cap alpha.. vs EBGD..cap alpha.. -Ab system, the 50% intercept was 3.25 +/- 0.32 ng for EBGD..cap alpha.. and 472.43 +/- 30.26 ng for EBGD..beta.. (p < 0.01), whereas, in the /sup 125/I-EBGD..beta.. vs EBGD..beta..-Ab system the 50% intercept was 3.51 +/- 0.58 for EBGD..beta.. and 166.77 +/- 49.20 for EBGD..cap alpha.. (p < 0.01). No significant differences were observed in the slopes of the dose response curves. It is concluded that the two subunits of ZP3 possess distinct immunologic characteristics as evaluated by RIA.« less

Bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects.

PubMed

Shah, Naman B; Duncan, Thomas M

2014-02-18

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit ε with the catalytic complex of Escherichia coli ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit ε could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of ε undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of ε's conformations is predominant. The assay measures kinetics of ε's binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound ε to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on ε's conformational changes.

Morphological modification of alpha-MnO2 catalyst for use in Li/air batteries.

PubMed

Park, Min-Sik; Kim, Jae-Hun; Kim, Ki Jae; Jeong, Goojin; Kim, Young-Jun

2013-05-01

Single crystal alpha-MnO2 nanowires and nanopowders have been successfully synthesized in order to facilitate a comparison of their catalytic activity for use in Li-air batteries. The importance of the morphological modification of the alpha-MnO2 catalyst for facilitating electrochemical reactions between Li and O2 is addressed. Distinctive catalytic activity of alpha-MnO2 is observed, which is in line with its different morphologies. The catalytic activity significantly affects the reversible capacity of Li-air batteries. A high aspect ratio, large surface area and good dispersibility of alpha-MnO2 in the nanowire form are advantageous providing larger active surfaces for promoting the fundamental reactions in Li-air batteries. We also introduce a robustly designed air-electrode composed of highly porous carbon and nanostructured alpha-MnO2 catalysts, with employs a metal foam current collector to ensure sufficient air-permeability and to maximize electronic conduction during cycles. Our suggestions should prove helpful in forming a basis for further investigations in developing advanced Li-air batteries.

Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass

PubMed Central

Lee, Young-Sam; Gregory, Mark T.; Yang, Wei

2014-01-01

DNA polymerase ζ (Pol ζ) is a eukaryotic B-family DNA polymerase that specializes in translesion synthesis and is essential for normal embryogenesis. At a minimum, Pol ζ consists of a catalytic subunit Rev3 and an accessory subunit Rev7. Mammalian Rev3 contains >3,000 residues and is twice as large as the yeast homolog. To date, no vertebrate Pol ζ has been purified for biochemical characterization. Here we report purification of a series of human Rev3 deletion constructs expressed in HEK293 cells and identification of a minimally catalytically active human Pol ζ variant. With a tagged form of an active Pol ζ variant, we isolated two additional accessory subunits of human Pol ζ, PolD2 and PolD3. The purified four-subunit Pol ζ4 (Rev3–Rev7–PolD2–PolD3) is much more efficient and more processive at bypassing a 1,2-intrastrand d(GpG)-cisplatin cross-link than the two-subunit Pol ζ2 (Rev3–Rev7). We show that complete bypass of cisplatin lesions requires Pol η to insert dCTP opposite the 3′ guanine and Pol ζ4 to extend the primers. PMID:24449906

Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass.

PubMed

Lee, Young-Sam; Gregory, Mark T; Yang, Wei

2014-02-25

DNA polymerase ζ (Pol ζ) is a eukaryotic B-family DNA polymerase that specializes in translesion synthesis and is essential for normal embryogenesis. At a minimum, Pol ζ consists of a catalytic subunit Rev3 and an accessory subunit Rev7. Mammalian Rev3 contains >3,000 residues and is twice as large as the yeast homolog. To date, no vertebrate Pol ζ has been purified for biochemical characterization. Here we report purification of a series of human Rev3 deletion constructs expressed in HEK293 cells and identification of a minimally catalytically active human Pol ζ variant. With a tagged form of an active Pol ζ variant, we isolated two additional accessory subunits of human Pol ζ, PolD2 and PolD3. The purified four-subunit Pol ζ4 (Rev3-Rev7-PolD2-PolD3) is much more efficient and more processive at bypassing a 1,2-intrastrand d(GpG)-cisplatin cross-link than the two-subunit Pol ζ2 (Rev3-Rev7). We show that complete bypass of cisplatin lesions requires Pol η to insert dCTP opposite the 3' guanine and Pol ζ4 to extend the primers.

Mapping of the immunophilin-immunosuppressant site of interaction on calcineurin.

PubMed

Husi, H; Luyten, M A; Zurini, M G

1994-05-13

The interaction of the immunosuppressive complexes cyclosporin A-cyclophilin A and FK506 binding protein-FK506 with the Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin has been investigated by means of photoaffinity labeling and chemical cross-linking. Photolabeling of purified bovine brain calcineurin with the affinity label [O-[4-[4-(1-diazo-2,2,2-trifluoroethyl)benzoyl]aminobutanoyl]-D- serine8]cyclosporin in the presence of cyclophilin A results, in addition to the labeling of cyclophilin itself, in the transfer of some of the chemical probe to both the catalytic subunit A and the regulatory subunit B of calcineurin. Chemical cross-linking studies with disuccinimidyl suberate in the presence of either cyclophilin A, B, or C in complex with cyclosporin A or FK506 binding protein-FK506 result on the other hand in the apparently exclusive and strictly immunosuppressant-dependent formation of covalent immunophilin-calcineurin B subunit products. Cross-linking of immunophilins to calcineurin B subunit requires the presence of subunit A. In the present study, using a set of recombinant maltose-binding protein fusion products representing different stretches of the catalytic subunit A, we were able to map the minimal calcineurin A sequence necessary for immunophilin-ligand-calcineurin B interaction to occur.

DOE Office of Scientific and Technical Information (OSTI.GOV)

McInnis, Brittney; Mitchell, Jessica; Marcus, Stevan, E-mail: smarcus@bama.ua.edu

Research highlights: {yields} cAMP deficiency induces phosphorylation of PKA catalytic subunit (Pka1) in S. pombe. {yields} Pka1 phosphorylation is further induced by physiological stresses. {yields} Pka1 phosphorylation is not induced in cells lacking the PKA regulatory subunit. {yields} Results suggest that cAMP-independent Pka1 phosphorylation is stimulatory in nature. -- Abstract: In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly highermore » levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1{Delta} cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1{Delta} cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1{sup +} or cyr1{Delta} S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.« less

Expression of membrane-bound and cytosolic guanylyl cyclases in the rat inner ear.

PubMed

Seebacher, T; Beitz, E; Kumagami, H; Wild, K; Ruppersberg, J P; Schultz, J E

1999-01-01

Membrane-bound guanylyl cyclases (GCs) are peptide hormone receptors whereas the cytosolic isoforms are receptors for nitric oxide. In the inner ear, the membrane-bound GCs may be involved in the regulation of fluid homeostasis and the cytosolic forms possibly play a role in signal processing and regulation of local blood flow. In this comprehensive study, we examined, qualitatively and quantitatively, the transcription pattern of all known GC isoforms in the inner ear from rat by RT-PCR. The tissues used were endolymphatic sac, stria vascularis, organ of Corti, organ of Corti outer hair cells, cochlear nerve, Reissner's membrane, vestibular dark cells, and vestibular sensory cells. We show that multiple particulate (GC-A, GC-B, GC-D, GC-E, GC-F and GC-G) and several subunits of the heterodimeric cytosolic GCs (alpha1, alpha2, beta1 and beta2) are expressed, albeit at highly different levels. GC-C was not found. GC-A and the soluble subunits alpha1 and beta1 were transcribed ubiquitously. GC-B was present in all tissues except stria vascularis, which contained GC-A and traces of GC-E and GC-G. GC-B was by far the predominant membrane-bound isoform in the organ of Corti (86%), Reissner's membrane (75%) and the vestibulum (80%). Surprisingly, GC-E, a retinal isoform, was detected in significant amounts in the cochlear nerve (8%) and in the organ of Corti (4%). Although the cytosolic GC is a heterodimer composed of an alpha and a beta subunit, the mRNA transcription of these subunits was not stoichiometric. Particularly in the vestibulum, the transcription of the beta1 subunits was at least four-fold higher than of the alpha1 subunit. The data are compatible with earlier suggestions that membrane receptor GCs may be involved in the control of inner ear electrolyte and fluid composition whereas NO-stimulated GC isoforms mainly participate in the regulation of blood flow and supporting cell physiology.

A heterobimetallic Ga/Yb-Schiff base complex for catalytic asymmetric alpha-addition of isocyanides to aldehydes.

PubMed

Mihara, Hisashi; Xu, Yingjie; Shepherd, Nicholas E; Matsunaga, Shigeki; Shibasaki, Masakatsu

2009-06-24

Development of a new heterobimetallic Ga(O-iPr)(3)/Yb(OTf)(3)/Schiff base 2d complex for catalytic asymmetric alpha-additions of isocyanides to aldehydes is described. Schiff base 2d derived from o-vanillin was suitable to utilize cationic rare earth metal triflates with good Lewis acidity in bimetallic Schiff base catalysis. The Ga(O-iPr)(3)/Yb(OTf)(3)/Schiff base 2d complex promoted asymmetric alpha-additions of alpha-isocyanoacetamides to aryl, heteroaryl, alkenyl, and alkyl aldehydes in good to excellent enantioselectivity (88-98% ee).

The alpha subunit of the epithelial sodium channel in the mouse: developmental regulation of its expression.

PubMed

Dagenais, A; Kothary, R; Berthiaume, Y

1997-09-01

Sodium reabsorption by the amiloride-sensitive sodium channel of epithelial cells plays a crucial role in the management of ionic composition and fluid volume in the body. In the respiratory system, sodium transport is involved in the clearance of pulmonary edema and of liquid secreted during fetal life at birth. We have cloned a partial cDNA of the alpha subunit of the mouse amiloride-sensitive sodium channel (alpha mENaC). In the region of comparison, the mouse alpha subunit shows 92% identity at the DNA level and 95% identity at the amino acid level with the rat sequence. The kidneys, lungs, and distal colon are major sites of expression of a 3.5-kb alpha mENaC mRNA. During mouse development, alpha mENaC transcripts appear late during gestation (d 17.5) and are expressed continuously thereafter. In the distal colon, a short 1.2-kb mRNA deleted of the 5' part of the transcript is detected during gestation and is replaced gradually by the mature 3.5-kb transcript after birth. Alpha mENaC and alpha1 Na+-K+-ATPase mRNAs have an expression profile that is modulated similarly during development for a given tissue. The expression of alpha mENaC transcripts increases transiently in the lungs at birth (2.5-fold), as for alpha1 Na+-K+-ATPase mRNAs (1.5-fold), suggesting that the expression of several components of the sodium transport system is modulated in the lungs at that time. In the kidney, there is no significant increase of alpha mENaC and alpha1 Na+-K+-ATPase mRNAs in newborns.

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bidart, J.M.; Troalen, F.; Salesse, R.

1987-06-25

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptidesmore » spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.« less

Multiple isoforms for the catalytic subunit of PKA in the basal fungal lineage Mucor circinelloides.

PubMed

Fernández Núñez, Lucas; Ocampo, Josefina; Gottlieb, Alexandra M; Rossi, Silvia; Moreno, Silvia

2016-12-01

Protein kinase A (PKA) activity is involved in dimorphism of the basal fungal lineage Mucor. From the recently sequenced genome of Mucor circinelloides we could predict ten catalytic subunits of PKA. From sequence alignment and structural prediction we conclude that the catalytic core of the isoforms is conserved, and the difference between them resides in their amino termini. This high number of isoforms is maintained in the subdivision Mucoromycotina. Each paralogue, when compared to the ones form other fungi is more homologous to one of its orthologs than to its paralogs. All of these fungal isoforms cannot be included in the class I or II in which fungal protein kinases have been classified. mRNA levels for each isoform were measured during aerobic and anaerobic growth. The expression of each isoform is differential and associated to a particular growth stage. We reanalyzed the sequence of PKAC (GI 20218944), the only cloned sequence available until now for a catalytic subunit of M. circinelloides. PKAC cannot be classified as a PKA because of its difference in the conserved C-tail; it shares with PKB a conserved C2 domain in the N-terminus. No catalytic activity could be measured for this protein nor predicted bioinformatically. It can thus be classified as a pseudokinase. Its importance can not be underestimated since it is expressed at the mRNA level in different stages of growth, and its deletion is lethal. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Comprehensive Characterization of AMP-activated Protein Kinase Catalytic Domain by Top-down Mass Spectrometry

PubMed Central

Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

2015-01-01

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ. C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ has noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems. PMID:26489410

Ligand-independent pathway that controls stability of interferon alpha receptor

PubMed Central

Liu, Jianghuai; Plotnikov, Alexander; Banerjee, Anamika; Kumar, K.G. Suresh; Ragimbeau, Josiane; Marijanovic, Zrinka; Baker, Darren P.; Pellegrini, Sandra; Fuchs, Serge Y.

2008-01-01

SUMMARY Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance. PMID:18166147

Binding of adenine to Stx2, the protein toxin from Escherichia coli O157:H7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fraser, Marie E., E-mail: frasm@ucalgary.ca; Cherney, Maia M.; Marcato, Paola

2006-07-01

Crystals of Stx2 were grown in the presence of adenosine and adenine. In both cases, the resulting electron density showed only adenine bound at the active site of the A subunit, proving that the holotoxin is an active N-glycosidase. Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active asmore » an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to ∼1.8 Å and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA.« less

Mutations in the PCCA gene encoding the {alpha} subunit of propionyl-CoA carboxylase in patients with propionic acidemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campeau, E.; Leon-Del-Rio, A.; Gravel, R.A.

Propionic acidemia is a rare autosomal recessive disorder characterized by a deficiency of the mitochondrial biotin-dependent enzyme, propionyl-CoA carboxylase (PCC). PCC has the structure {alpha}{sub 4}{beta}{sub 4}, with the {alpha} subunit containing the biotin prosthetic group. This study is concerned with defining the spectrum of mutations occurring in the PCCA gene encoding the {alpha} subunit. Mutations were initially assigned to this gene through complementation experiments done after somatic fusion of patient fibroblasts. The analyses were performed on PCR-amplified reverse transcripts of fibroblast RNA. The mutations were identified by single strand conformational polymorphism analysis and direct sequencing of PCR products. Threemore » candidate disease-causing mutations and one DNA polymorphism were identified in the {alpha} subunit sequence in different patients: (1) a 3 bp deletion {triangle}CTG{sub 2058-2060}, which eliminates Cys687 near the biotin binding site (Lys669); (2) T{sub 611}{r_arrow}A which converts Met204 to Lys in a highly conserved region matching that of an ATP binding site; (3) An {approximately}50 bp deletion near the 3{prime} end of the cDNA which likely corresponds to the loss of an exon due to a splicing defect; and (4) a 3 bp insertion, +CAG{sub 2203}, located downstream of the stop codon, which is likely a DNA polymorphism. In order to determine the effect of the Cys687 deletion on the biotinylation of PCC, we expressed the mutation in a 67 amino acid C-terminal fragment of the PCC {alpha} subunit in E. coli in which biotinylation is directed by the bacterial biotin ligase. While the mutant peptide was expressed at about half-normal levels, the biotinylation of the peptide that was present was reduced to only {approximately}20% normal. We suggest, therefore, that the absence of PCC activity due to {triangle}Cys687 results at least in part from defective biotinylation of an unstable protein.« less

The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I

PubMed Central

1995-01-01

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response protein. PMID:7860628

Dicamba Monooxygenase: Structural Insights into a Dynamic Rieske Oxygenase that Catalyzes an Exocyclic Monooxygenation

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Ordine, Robert L.; Rydel, Timothy J.; Storek, Michael J.

2009-09-08

Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O{sub 2} into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer ({alpha}{sub 3}) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While themore » Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co{sup 2+}, which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 {angstrom}, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.« less

Spacing requirements for interactions between the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase and the cAMP receptor protein.

PubMed Central

Lloyd, G S; Busby, S J; Savery, N J

1998-01-01

During transcription initiation at bacterial promoters, the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) can interact with DNA-sequence elements (known as UP elements) and with activator proteins. We have constructed a series of semi-synthetic promoters carrying both an UP element and a consensus DNA-binding site for the Escherichia coli cAMP receptor protein (CRP; a factor that activates transcription by making direct contacts with alphaCTD). At these promoters, the UP element was located at a variety of distances upstream of the CRP-binding site, which was fixed at position -41.5 bp upstream of the transcript start. At some positions, the UP element caused enhanced promoter activity whereas, at other positions, it had very little effect. In no case was the CRP-dependence of the promoter relieved. DNase I and hydroxyl-radical footprinting were used to study ternary RNA polymerase-CRP-promoter complexes formed at two of the most active of these promoters, and co-operativity between the binding of CRP and purified alpha subunits was studied. The footprints show that alphaCTD binds to the UP element as it is displaced upstream but that this displacement does not prevent alphaCTD from being contacted by CRP. Models to account for this are discussed. PMID:9461538

Protein Kinase CK2 Regulates Cytoskeletal Reorganization during Ionizing Radiation-Induced Senescence of Human Mesenchymal Stem Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Daojing; Jang, Deok-Jin

2009-08-21

Human mesenchymal stem cells (hMSC) are critical for tissue regeneration. How hMSC respond to genotoxic stresses and potentially contribute to aging and cancer remain underexplored. We demonstrated that ionizing radiation induced cellular senescence of hMSC over a period of 10 days, showing a critical transition between day 3 and day 6. This was confirmed by senescence-associated beta-galactosidase (SA-{beta}-gal) staining, protein expression profiles of key cell cycle regulators (retinoblastoma (Rb) protein, p53, p21{sup waf1/Cip1}, and p16{sup INK4A}), and senescence-associated secretory phenotypes (SASPs) (IL-8, IL-12, GRO, and MDC). We observed dramatic cytoskeletal reorganization of hMSC through reduction of myosin-10, redistribution of myosin-9,more » and secretion of profilin-1. Using a SILAC-based phosphoproteomics method, we detected significant reduction of myosin-9 phosphorylation at Ser1943, coinciding with its redistribution. Importantly, through treatment with cell permeable inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT)), and gene knockdown using RNA interference, we identified CK2, a kinase responsible for myosin-9 phosphorylation at Ser1943, as a key factor contributing to the radiation-induced senescence of hMSC. We showed that individual knockdown of CK2 catalytic subunits CK2{alpha} and CK2{alpha}{prime} induced hMSC senescence. However, only knockdown of CK2{alpha} resulted in morphological phenotypes resembling those of radiation-induced senescence. These results suggest that CK2{alpha} and CK2{alpha}{prime} play differential roles in hMSC senescence progression, and their relative expression might represent a novel regulatory mechanism for CK2 activity.« less

High concentration of antioxidants N-acetylcysteine and mitoquinone-Q induces intercellular adhesion molecule 1 and oxidative stress by increasing intracellular glutathione.

PubMed

Mukherjee, Tapan K; Mishra, Anurag K; Mukhopadhyay, Srirupa; Hoidal, John R

2007-02-01

In endothelial cells, the intracellular level of glutathione is depleted during offering protection against proinflammatory cytokine TNF-alpha-induced oxidative stress. Administration of anti-inflammatory drugs, i.e., N-acetylcysteine (NAC) or mitoquinone-Q (mito-Q) in low concentrations in the human pulmonary aortic endothelial cells offered protection against depletion of reduced glutathione and oxidative stress mediated by TNF-alpha. However, this study addressed that administration of NAC or mito-Q in high concentrations resulted in a biphasic response by initiating an enhanced generation of both reduced glutathione and oxidized glutathione and enhanced production of reactive oxygen species, along with carbonylation and glutathionylation of the cellular proteins. This study further addressed that IkappaB kinase (IKK), a phosphorylation-dependent regulator of NF-kappaB, plays an important regulatory role in the TNF-alpha-mediated induction of the inflammatory cell surface molecule ICAM-1. Of the two catalytic subunits of IKK (IKKalpha and IKKbeta), low concentrations of NAC and mito-Q activated IKKalpha activity, thereby inhibiting the downstream NF-kappaB and ICAM-1 induction by TNF-alpha. High concentrations of NAC and mito-Q instead caused glutathionylation of IKKalpha, thereby inhibiting its activity that in turn enhanced the downstream NF-kappaB activation and ICAM-1 expression by TNF-alpha. Thus, establishing IKKalpha as an anti-inflammatory molecule in endothelial cells is another focus of this study. This is the first report that describes a stressful situation in the endothelial cells created by excess of antioxidative and anti-inflammatory agents NAC and mito-Q, resulting in the generation of reactive oxygen species, carbonylation and glutathionylation of cellular proteins, inhibition of IKKalpha activity, and up-regulation of ICAM-1expression.

Solution structure of {alpha}-conotoxin PIA, a novel antagonist of {alpha}6 subunit containing nicotinic acetylcholine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chi, Seung-Wook; Lee, Si-Hyung; Kim, Do-Hyoung

2005-12-30

{alpha}-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing {alpha}6 and {alpha}3 subunits. {alpha}-conotoxin PIA displays 75-fold higher affinity for rat {alpha}6/{alpha}3{beta}2{beta}3 nAChRs than for rat {alpha}3{beta}2 nAChRs. We have determined the three-dimensional structure of {alpha}-conotoxin PIA by nuclear magnetic resonance spectroscopy. The {alpha}-conotoxin PIA has an '{omega}-shaped' overall topology as other {alpha}4/7 subfamily conotoxins. Yet, unlike other neuronally targeted {alpha}4/7-conotoxins, its N-terminal tail Arg{sup 1}-Asp{sup 2}-Pro{sup 3} protrudes out of its main molecular body because Asp{sup 2}-Pro{sup 3}-Cys{sup 4}-Cys{sup 5} forms a stable type I {beta}-turn. In addition, amore » kink introduced by Pro{sup 15} in the second loop of this toxin provides a distinct steric and electrostatic environment from those in {alpha}-conotoxins MII and GIC. By comparing the structure of {alpha}-conotoxin PIA with other functionally related {alpha}-conotoxins we suggest structural features in {alpha}-conotoxin PIA that may be associated with its unique receptor recognition profile.« less

Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants.

PubMed

Xie, Gary; Forst, Christian; Bonner, Carol; Jensen, Roy A

2002-01-01

Tryptophan synthase consists of two subunits, alpha and beta. Two distinct subgroups of beta chain exist. The major group (TrpEb_1) includes the well-studied beta chain of Salmonella typhimurium. The minor group of beta chain (TrpEb_2) is most frequently found in the Archaea. Most of the amino-acid residues important for catalysis are highly conserved between both TrpE subfamilies. Conserved amino-acid residues of TrpEb_1 that make allosteric contact with the TrpEa subunit (the alpha chain) are absent in TrpEb_2. Representatives of Archaea, Bacteria and higher plants all exist that possess both TrpEb_1 and TrpEb_2. In those prokaryotes where two trpEb genes coexist, one is usually trpEb_1 and is adjacent to trpEa, whereas the second is trpEb_2 and is usually unlinked with other tryptophan-pathway genes. TrpEb_1 is nearly always partnered with TrpEa in the tryptophan synthase reaction. However, by default at least six lineages of the Archaea are likely to use TrpEb_2 as the functional beta chain, as TrpEb_1 is absent. The six lineages show a distinctive divergence within the overall TrpEa phylogenetic tree, consistent with the lack of selection for amino-acid residues in TrpEa that are otherwise conserved for interfacing with TrpEb_1. We suggest that the standalone function of TrpEb_2 might be to catalyze the serine deaminase reaction, an established catalytic capability of tryptophan synthase beta chains. A coincident finding of interest is that the Archaea seem to use the citramalate pathway, rather than threonine deaminase (IlvA), to initiate the pathway of isoleucine biosynthesis.

Alpha4 containing nicotinic receptors are positioned to mediate postsynaptic effects on serotonin neurons in the rat dorsal raphe nucleus

PubMed Central

Commons, Kathryn G.

2008-01-01

Nicotinic acetylcholine receptors containing the alpha4 and beta2 subunits constitute the most abundant high-affinity binding site of nicotine in the brain and are critical for the addictive qualities of nicotine. Serotonin neurotransmission is thought to be an important contributor to nicotine addiction. Therefore in this study it was examined how alpha4-containing receptors are positioned to modulate the function of serotonin neurons using ultrastructural analysis of immunolabeling for the alpha4 receptor subunit in the dorsal raphe nucleus (DR), a primary source of forebrain serotonin in the rat. Of 150 profiles labeled for the alpha4 subunit, 140 or 93% consisted of either soma or dendrites, these were often small-caliber (distal) dendrites <1.5 um in diameter (63/150 or 42%). The majority (107/150 or 71%) of profiles containing labeling for alpha4 were dually labeled for the synthetic enzyme for serotonin, tryptophan hydroxylase (TPH). Within dendrites immunogold labeling for alpha4 was present on the plasma membrane or near postsynaptic densities. However, labeling for alpha4 was commonly localized to the cytoplasmic compartment often associated with smooth endoplasmic reticulum, plausibly representing receptors in transit to or from the plasma membrane. Previous studies have suggested that nicotine presynaptically regulates activity onto serotonin neurons, however alpha4 immunolabeling was detected in only 10 axons in the DR or 7% of profiles sampled. This finding suggest that alpha4 containing receptors are minor contributors to presynaptic regulation of synaptic activity onto serotonin neurons, but rather alpha4 containing receptors are positioned to influence serotonin neurons directly at postsynaptic sites. PMID:18403129

Steady-state levels of G-protein beta-subunit expression are regulated by treatment of cells with bacterial toxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watkins, D.C.; Northup, J.K.; Malbon, C.C.

1987-05-01

Cultures of 3T3-L1 cells were incubated with either 10 ng/ml cholera toxin or 10 ng/ml pertussis toxin from 4 days prior to the initiation of differentiation and throughout the subsequent incubation. Toxin concentrations were sufficient to completely prevent the labelling of alpha-subunits with (/sup 32/P)NAD/sup +/ and pertussis toxin and to prevent by more than 90% the labelling with (/sup 32/P)NAD/sup +/ and cholera toxin in membranes prepared from these cells. Neither toxin prevented the differentiation to the adipocyte phenotype. Neither toxin prevented the increases in the relative amounts of G-proteins which occur upon differentiation. Both toxins dramatically decreased themore » amount of beta-subunits. As measured by quantitative immunoblotting with antisera specific for both the 35 kDa and 36 kDa beta-subunits, levels of beta-subunit were decreased by more than 50% of steady-state level of control cells. Thus, bacterial toxins which modifies G-protein alpha-subunits are capable of modulating the levels of beta-subunits in vivo. The basis for the regulation of G-protein subunit expression by bacterial toxins is under study.« less

Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

PubMed

Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

1994-10-25

DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1.

Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

PubMed Central

Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

1994-01-01

DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1. Images PMID:7971267

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.

PubMed Central

Geib, Sandrine; Sandoz, Guillaume; Mabrouk, Kamel; Matavel, Alessandra; Marchot, Pascale; Hoshi, Toshinori; Villaz, Michel; Ronjat, Michel; Miquelis, Raymond; Lévêque, Christian; de Waard, Michel

2002-01-01

Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit. PMID:11988102

Oxygen binding by alpha(Fe2+)2beta(Ni2+)2 hemoglobin crystals.

PubMed Central

Bruno, S.; Bettati, S.; Manfredini, M.; Mozzarelli, A.; Bolognesi, M.; Deriu, D.; Rosano, C.; Tsuneshige, A.; Yonetani, T.; Henry, E. R.

2000-01-01

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of alpha and beta subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of alpha and beta hemes separately. Previous studies have characterized the oxygen binding by alpha(Ni2+)2beta(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of alpha(Fe2+)2beta(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 +/- 3 and 87 +/- 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 +/- 0.06 and 0.90 +/- 0.03. Analysis of the binding curves, taking into account the different projections of the alpha hemes along the optical directions, indicates that the oxygen affinity of alpha1 hemes is 1.3-fold lower than alpha2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the beta subunits of alpha(Ni2+)2beta(Fe2+)2 crystals. The average oxygen affinity of the alpha subunits (p50 = 91 Torr) is about 1.2-fold higher than the beta subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model. PMID:10794410

Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein.

PubMed Central

Ayers, D J; Sunshine, M G; Six, E W; Christie, G E

1994-01-01

The bacteriophage P2 ogr gene product is a positive regulator of transcription from P2 late promoters. The ogr gene was originally defined by compensatory mutations that overcame the block to P2 growth imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of RNA polymerase. DNA sequence analysis has confirmed that this mutation affects the C-terminal region of the alpha subunit, changing a leucine residue at position 290 to a histidine (rpoAL290H). We have employed a reporter plasmid system to screen other, previously described, rpoA mutants for effects on activation of a P2 late promoter and have identified a second allele, rpoA155, that blocks P2 late transcription. This mutation lies just upstream of rpoAL290H, changing the leucine residue at position 289 to a phenylalanine (rpoAL289F). The effect of the rpoAL289F mutation is not suppressed by the rpoAL290H-compensatory P2 ogr mutation. P2 ogr mutants that overcome the block imposed by rpoAL289F were isolated and characterized. Our results are consistent with a direct interaction between Ogr and the alpha subunit of RNA polymerase and support a model in which transcription factor contact sites within the C terminus of alpha are discrete and tightly clustered. PMID:8002564

Uncovering the Role of Sgf73 in Maintaining SAGA Deubiquitinating Module Structure and Activity

DOE PAGES

Yan, Ming; Wolberger, Cynthia

2014-12-17

The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex performs multiple functions in transcription activation including deubiquitinating histone H2B, which is mediated by a subcomplex called the deubiquitinating module (DUBm). The yeast DUBm comprises a catalytic subunit, Ubp8, and three additional subunits, Sgf11, Sus1 and Sgf73, all of which are required for DUBm activity. A portion of the non-globular Sgf73 subunit lies between the Ubp8 catalytic domain and the ZnF-UBP domain and has been proposed to contribute to deubiquitinating activity by maintaining the catalytic domain in an active conformation. We report structural and solution studies of the DUBm containing two different Sgf73 point mutations thatmore » disrupt deubiquitinating activity. We find that the Sgf73 mutations abrogate deubiquitinating activity by impacting the Ubp8 ubiquitin-binding fingers region and have an unexpected effect on the overall folding and stability of the DUBm complex. Finally, taken together, our data suggest a role for Sgf73 in maintaining both the organization and ubiquitin-binding conformation of Ubp8, thereby contributing to overall DUBm activity.« less

Actin-related proteins regulate the RSC chromatin remodeler by weakening intramolecular interactions of the Sth1 ATPase.

PubMed

Turegun, Bengi; Baker, Richard W; Leschziner, Andres E; Dominguez, Roberto

2018-01-01

The catalytic subunits of SWI/SNF-family and INO80-family chromatin remodelers bind actin and actin-related proteins (Arps) through an N-terminal helicase/SANT-associated (HSA) domain. Between the HSA and ATPase domains lies a conserved post-HSA (pHSA) domain. The HSA domain of Sth1, the catalytic subunit of the yeast SWI/SNF-family remodeler RSC, recruits the Rtt102-Arp7/9 heterotrimer. Rtt102-Arp7/9 regulates RSC function, but the mechanism is unclear. We show that the pHSA domain interacts directly with another conserved region of the catalytic subunit, protrusion-1. Rtt102-Arp7/9 binding to the HSA domain weakens this interaction and promotes the formation of stable, monodisperse complexes with DNA and nucleosomes. A crystal structure of Rtt102-Arp7/9 shows that ATP binds to Arp7 but not Arp9. However, Arp7 does not hydrolyze ATP. Together, the results suggest that Rtt102 and ATP stabilize a conformation of Arp7/9 that potentiates binding to the HSA domain, which releases intramolecular interactions within Sth1 and controls DNA and nucleosome binding.

Scaleable catalytic asymmetric Strecker syntheses of unnatural alpha-amino acids.

PubMed

Zuend, Stephan J; Coughlin, Matthew P; Lalonde, Mathieu P; Jacobsen, Eric N

2009-10-15

Alpha-amino acids are the building blocks of proteins and are widely used as components of medicinally active molecules and chiral catalysts. Efficient chemo-enzymatic methods for the synthesis of enantioenriched alpha-amino acids have been developed, but it is still a challenge to obtain non-natural amino acids. Alkene hydrogenation is broadly useful for the enantioselective catalytic synthesis of many classes of amino acids, but it is not possible to obtain alpha-amino acids bearing aryl or quaternary alkyl alpha-substituents using this method. The Strecker synthesis-the reaction of an imine or imine equivalent with hydrogen cyanide, followed by nitrile hydrolysis-is an especially versatile chemical method for the synthesis of racemic alpha-amino acids. Asymmetric Strecker syntheses using stoichiometric amounts of a chiral reagent have been applied successfully on gram-to-kilogram scales, yielding enantiomerically enriched alpha-amino acids. In principle, Strecker syntheses employing sub-stoichiometric quantities of a chiral reagent could provide a practical alternative to these approaches, but the reported catalytic asymmetric methods have seen limited use on preparative scales (more than a gram). The limited utility of existing catalytic methods may be due to several important factors, including the relatively complex and precious nature of the catalysts and the requisite use of hazardous cyanide sources. Here we report a new catalytic asymmetric method for the syntheses of highly enantiomerically enriched non-natural amino acids using a simple chiral amido-thiourea catalyst to control the key hydrocyanation step. This catalyst is robust, without sensitive functional groups, so it is compatible with aqueous cyanide salts, which are safer and easier to handle than other cyanide sources; this makes the method adaptable to large-scale synthesis. We have used this new method to obtain enantiopure amino acids that are not readily prepared by enzymatic methods or by chemical hydrogenation.

Spectral and Temporal Properties of the Alpha and Beta Subunits and (alpha Beta) Monomer Isolated from Nostoc SP. Using Picosecond Laser Spectroscopy.

NASA Astrophysics Data System (ADS)

Dagen, Aaron J.

1985-12-01

The fluorescence decay profiles, relative quantum yield and transmission of the (alpha), (beta) and ((alpha)(beta)) complexes from phycoerythrin isolated from the photosynthetic antenna system of Nostoc sp. and measured by single picosecond laser spectroscopic techniques is studied. The fluorescence decay profiles of all three complexes are found to be intensity independent for the intensity range investigated ((TURN)4 x 10('13) to (TURN)4 x 10('15) photons-cm('-2) per pulse). The apparent decrease in the relative quantum yield of all three complexes as intensity increases is offset by a corresponding increase in the relative transmission. This evidence, along with the intensity independent fluorescence kinetics, suggests that exciton annihilation is absent in these complexes. The decay profiles are fit to models assuming energy transfer amongst fluorescing chromophores. The intraprotein transfer rate is found to be 100 ps in the (alpha) subunit, 666 ps in the (beta) subunit. Constraining these rates to be identical in the monomer results in explaining the monomer kinetics by an increase in the nonradiative rate of the f(,(beta)) chromophore, an apparent result of aggregation effects.

Spectral and temporal properties of the alpha and beta subunits and (alpha beta) monomer isolated from Nostoc sp. using picosecond laser spectroscopy

NASA Astrophysics Data System (ADS)

Dagen, A. J.

1985-12-01

The fluorescence decay profiles, relative quantum yield and transmission of the alpha, beta and (alpha beta) complexes from phycoerythrin isolated from the photosynthetic antenna system of Nostoc sp. and measured by single picosecond laser spectroscopic techniques is studied. The fluorescence decay profiles of all three complexes are found to be intensity independent for the intensity range investigated (approx. 4x10 to the 13th power to 4x10 to the 15th power photons/sq cm per pulse). The apparent decrease in the relative quantum yield of all three complexes as intensity increases is offset by a corresponding increase in the relative transmission. This evidence, along with the intensity independent fluorescence kinetics, suggests that exciton annihilation is absent in these complexes. The decay profiles are fit to models assuming energy transfer amongst fluorescing chromophores. The intraprotein transfer rate is found to be 100 ps in the alpha subunit, 666 ps in the beta subunit. Constraining these rates to be identical in the monomer results in explaining the monomer kinetics by an increase in the nonradiative rate of the f beta chromophore, an apparent result of aggregation effects.

Nicotinic receptor abnormalities in the cerebellar cortex in autism.

PubMed

Lee, M; Martin-Ruiz, C; Graham, A; Court, J; Jaros, E; Perry, R; Iversen, P; Bauman, M; Perry, E

2002-07-01

Autism is a common developmental disorder associated with structural and inferred neurochemical abnormalities of the brain. Cerebellar abnormalities frequently have been identified, based on neuroimaging or neuropathology. Recently, the cholinergic neurotransmitter system has been implicated on the basis of nicotinic receptor loss in the cerebral cortex. Cerebellar cholinergic activities were therefore investigated in autopsy tissue from a series of autistic individuals. The presynaptic cholinergic enzyme, choline acetyltransferase, together with nicotinic and muscarinic receptor subtypes were compared in the cerebellum from age-matched mentally retarded autistic (eight), normal control (10) and non-autistic mentally retarded individuals (11). The nicotinic receptor binding the agonist epibatidine (the high affinity receptor subtype, consisting primarily of alpha3 and alpha4, together with beta2 receptor subunits) was significantly reduced by 40-50% in the granule cell, Purkinje and molecular layers in the autistic compared with the normal group (P < 0.05). There was an opposite increase (3-fold) in the nicotinic receptor binding alpha-bungarotoxin (to the alpha7 subunit) which reached significance in the granule cell layer (P < 0.05). These receptor changes were paralleled by a significant reduction (P < 0.05) and non-significant increase, respectively, of alpha4 and alpha7 receptor subunit immunoreactivity measured using western blotting. Immunohistochemically loss of alpha(4 )reactivity was apparent from Purkinje and the other cell layers, with increased alpha7 reactivity in the granule cell layer. There were no significant changes in choline acetyltransferase activity, or in muscarinic M1 and M2 receptor subtypes in autism. In the non-autistic mentally retarded group, the only significant abnormality was a reduction in epibatidine binding in the granule cell and Purkinje layers. In two autistic cases examined histologically, Purkinje cell loss was observed in multiple lobules throughout the vermis and hemispheres. This was more severe in one case with epilepsy, which also showed vermis folial malformation. The case with less severe Purkinje cell loss also showed cerebellar white matter thinning and demyelination. These findings indicate a loss of the cerebellar nicotinic alpha4 receptor subunit in autism which may relate to the loss of Purkinje cells, and a compensatory increase in the alpha7 subunit. It remains to be determined how these receptor abnormalities are involved in neurodevelopment in autism and what is the relationship to mental function. Since nicotinic receptor agonists enhance attentional function and also induce an elevation in the high affinity receptor, nicotinic therapy in autism may be worth considering.

Altered expression of extracellular matrix molecules and their receptors in chronic pancreatitis and pancreatic adenocarcinoma in comparison with normal pancreas.

PubMed

Shimoyama, S; Gansauge, F; Gansauge, S; Oohara, T; Beger, H G

1995-12-01

The aim of this study was to elucidate the expression and distribution patterns of both integrins and extracellular matrix (ECM) molecules in chronic pancreatitis (CP) and pancreatic adenocarcinoma (PC) compared with normal pancreas (NP). Expression of nine alpha-subunits (alpha 2-alpha 6, alpha V, alpha L, alpha M, and alpha X), four beta-subunits (beta 1, beta 3-beta 5), and four ECM molecules (type IV collagen, laminin, fibronectin, and vitronectin) was investigated immunohistochemically. In CP, all integrins except alpha V showed nearly the same staining patterns compared with NP. Some acinar cells in CP expressed alpha V. Whereas alpha 2, alpha 3, and alpha 6 expression was stronger and diffuse, no alpha 5 expression was seen in PC. Basement membrane (BM) showed continuous staining in CP, whereas it showed discontinuous/absent staining in PC with antitype IV collagen, laminin, and vitronectin antibodies. Some carcinoma cells showed reverse correlation between alpha 2, alpha 3, and alpha 6 expression and type IV collagen and laminin expression. Fibronectin showed diffuse stromal expression in CP and PC. Some acinar cells or duct cells in CP carcinoma cells in PC showed intracellular VN expression. These results suggest that these integrins and ECM molecules are involved in inflammatory and malignant processes in pancreas.

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

PubMed

Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

1996-07-01

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

Elastase-like Activity Is Dominant to Chymotrypsin-like Activity in 20S Proteasome's β5 Catalytic Subunit.

PubMed

Bensinger, Dennis; Neumann, Theresa; Scholz, Christoph; Voss, Constantin; Knorr, Sabine; Kuckelkorn, Ulrike; Hamacher, Kay; Kloetzel, Peter-Michael; Schmidt, Boris

2016-07-15

The ubiquitin/proteasome system is the major protein degradation pathway in eukaryotes with several key catalytic cores. Targeting the β5 subunit with small-molecule inhibitors is an established therapeutic strategy for hematologic cancers. Herein, we report a mouse-trap-like conformational change that influences molecular recognition depending on the substitution pattern of a bound ligand. Variation of the size of P1 residues from the highly β5-selective proteasome inhibitor BSc2118 allows for discrimination between inhibitory strength and substrate conversion. We found that increasing molecular size strengthens inhibition, whereas decreasing P1 size accelerates substrate conversion. Evaluation of substrate hydrolysis after silencing of β5 activity reveals significant residual activity for large residues exclusively. Thus, classification of the β5 subunit as chymotrypsin-like and the use of the standard tyrosine-containing substrate should be reconsidered.

[beta]-Glucan Synthesis in the Cotton Fiber (III. Identification of UDP-Glucose-Binding Subunits of [beta]-Glucan Synthases by Photoaffinity Labeling with [[beta]-32P]5[prime]-N3-UDP-Glucose.

PubMed Central

Li, L.; Drake, R. R.; Clement, S.; Brown, R. M.

1993-01-01

Using differential product entrapment and photolabeling under specifying conditions, we identifIed a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Gossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring [beta]-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of [beta]-1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring [beta]-1,3-glucan synthesis, is coincident with [beta]-1,3-glucan synthase activity, and is calcium dependent. The possible involvement of other polypeptides in the synthesis of [beta]-1,3-glucan is discussed. PMID:12231766

Each Monomer of the Dimeric Accessory Protein for Human Mitochondrial DNA Polymerase Has a Distinct Role in Conferring Processivity*

PubMed Central

Lee, Young-Sam; Lee, Sujin; Demeler, Borries; Molineux, Ian J.; Johnson, Kenneth A.; Yin, Y. Whitney

2010-01-01

The accessory protein polymerase (pol) γB of the human mitochondrial DNA polymerase stimulates the synthetic activity of the catalytic subunit. pol γB functions by both accelerating the polymerization rate and enhancing polymerase-DNA interaction, thereby distinguishing itself from the accessory subunits of other DNA polymerases. The molecular basis for the unique functions of human pol γB lies in its dimeric structure, where the pol γB monomer proximal to pol γA in the holoenzyme strengthens the interaction with DNA, and the distal pol γB monomer accelerates the reaction rate. We further show that human pol γB exhibits a catalytic subunit- and substrate DNA-dependent dimerization. By duplicating the monomeric pol γB of lower eukaryotes, the dimeric mammalian proteins confer additional processivity to the holoenzyme polymerase. PMID:19858216

Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato.

PubMed Central

Millar, A H; Knorpp, C; Leaver, C J; Hill, S A

1998-01-01

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 micromol/min per mg of protein. The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were alpha subunits of pyruvate dehydrogenase, and the 37 kDa protein was the beta subunit of pyruvate dehydrogenase. N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein. Incubation of the mPDC with [2-14C]pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins. PMID:9729464

Studies of the Outer Membrane Proteins of Campylobacter Jejuni for Vaccine Development

DTIC Science & Technology

1991-11-26

Mycobacterium tuberculosis, and M.leprae (66%) and mitochondrial protein p1 precursor of human and Chinese hamster cells (64%), and rubisco subunit binding...175) SAWG--DIgNIISDAP’KXVGRXgVITVK (202) 64% Rubisco subunit binding-protein alpha subunit of wheat (151) SAGN--OELIZGANADAIDOGPOVVLStE (178) 57

Baicalin and scutellarin are proteasome inhibitors that specifically target chymotrypsin-like catalytic activity.

PubMed

Wu, Yi-Xin; Sato, Eiji; Kimura, Wataru; Miura, Naoyuki

2013-09-01

Baicalin and scutellarin are the major active principal flavonoids extracted from the Chinese herbal medicines Scutellaria baicalensis and Erigeron breviscapus (Vant.) Hand-Mazz. It has recently been reported that baicalin and scutellarin have antitumor activity. However, the mechanisms of action are unknown. We previously reported that some flavonoids have a specific role in the inhibition of the activity of proteasome subunits and induced apoptosis in tumor cells. To further investigate these pharmacological effects, we examined the inhibitory activity of baicalin and scutellarin on the extracted proteasomes from mice and cancer cells. Using fluorogenic substrates for proteasome catalytic subunits, we found that baicalin and scutellarin specifically inhibited chymotrypsin-like activity but did not inhibit trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities. These data suggested that baicalin and scutellarin specifically inhibit chymotrypsin-like catalytic activity in the proteasome. Copyright © 2012 John Wiley & Sons, Ltd.

ENU mutagenesis identifies mice with cardiac fibrosis and hepatic steatosis caused by a mutation in the mitochondrial trifunctional protein beta-subunit.

PubMed

Kao, Hsiao-Jung; Cheng, Ching-Feng; Chen, Yen-Hui; Hung, Shuen-Iu; Huang, Cheng-Chih; Millington, David; Kikuchi, Tateki; Wu, Jer-Yuarn; Chen, Yuan-Tsong

2006-12-15

Using the metabolomics-guided screening coupled to N-ethyl-N-nitrosourea-mediated mutagenesis, we identified mice that exhibited elevated levels of long-chain acylcarnitines. Whole genome homozygosity mapping with 262 SNP markers mapped the disease gene to chromosome 5 where candidate genes Hadha and Hadhb, encoding the mitochondria trifunctional protein (MTP) alpha- and beta-subunits, respectively, are located. Direct sequencing revealed a normal alpha-subunit, but detected a nucleotide T-to-A transversion in exon 14 (c.1210T>A) of beta-subunit (Hadhb) which resulted in a missense mutation of methionine to lysine (M404K). Western blot analysis showed a significant reduction of both the alpha- and beta-subunits, consistent with reduced enzyme activity in both the long-chain 3-hydroxyacyl-CoA dehydrogenase and the long-chain 3-ketoacyl-CoA thiolase activities. These mice had a decreased weight gain and cardiac arrhythmias which manifested from a prolonged PR interval to a complete atrio-ventricular dissociation, and died suddenly between 9 and 16 months of age. Histopathological studies showed multifocal cardiac fibrosis and hepatic steatosis. This mouse model will be useful to further investigate the mechanisms underlying arrhythmogenesis relating to lipotoxic cardiomyopathy and to investigate pathophysiology and treatment strategies for human MTP deficiency.

Astrocytes express specific variants of CaM KII delta and gamma, but not alpha and beta, that determine their cellular localizations.

PubMed

Vallano, M L; Beaman-Hall, C M; Mathur, A; Chen, Q

2000-04-01

Multiple isoforms of type II Ca(2+)-calmodulin-dependent kinase (CaM KII) are composed of two major neuron-specific subunits, designated alpha and beta, and two less well-characterized subunits that are also expressed in non-neuronal tissues, designated delta and gamma. Regulated expression of these 4 gene products, and several variants produced by alternative splicing, shows temporal and regional specificity and influences intracellular targeting. We used immunoblotting and RT-PCR to analyze subunit and variant expression and distribution in cultured cerebellar astrocytes and neurons, and whole cerebellar cortex from rodent brain. The data indicate that: (i) astrocytes express a single splice variant of delta, namely delta(2); (ii) like neurons, astrocytes express two forms of CaM KII gamma; gamma(B) and gamma(A); (iii) these CaM KII variants are enriched in the supernate fraction in astrocytes, and the particulate fraction in neurons; (iv) unlike neurons, astrocytes do not express detectable levels of alpha or beta subunits or their respective splice variants. The results indicate that neurons and astrocytes express distinct CaM KII subunits and variants that localize to distinct subcellular compartments and, by inference, exert distinct cellular functions. Copyright 2000 Wiley-Liss, Inc.

B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability

PubMed Central

Houge, Gunnar; Haesen, Dorien; Vissers, Lisenka E.L.M.; Mehta, Sarju; Parker, Michael J.; Wright, Michael; Vogt, Julie; McKee, Shane; Tolmie, John L.; Cordeiro, Nuno; Kleefstra, Tjitske; Willemsen, Marjolein H.; Reijnders, Margot R.F.; Berland, Siren; Hayman, Eli; Lahat, Eli; Brilstra, Eva H.; van Gassen, Koen L.I.; Zonneveld-Huijssoon, Evelien; de Bie, Charlotte I.; Hoischen, Alexander; Eichler, Evan E.; Holdhus, Rita; Steen, Vidar M.; Døskeland, Stein Ove; Hurles, Matthew E.; FitzPatrick, David R.; Janssens, Veerle

2015-01-01

Here we report inherited dysregulation of protein phosphatase activity as a cause of intellectual disability (ID). De novo missense mutations in 2 subunits of serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) were identified in 16 individuals with mild to severe ID, long-lasting hypotonia, epileptic susceptibility, frontal bossing, mild hypertelorism, and downslanting palpebral fissures. PP2A comprises catalytic (C), scaffolding (A), and regulatory (B) subunits that determine subcellular anchoring, substrate specificity, and physiological function. Ten patients had mutations within a highly conserved acidic loop of the PPP2R5D-encoded B56δ regulatory subunit, with the same E198K mutation present in 6 individuals. Five patients had mutations in the PPP2R1A-encoded scaffolding Aα subunit, with the same R182W mutation in 3 individuals. Some Aα cases presented with large ventricles, causing macrocephaly and hydrocephalus suspicion, and all cases exhibited partial or complete corpus callosum agenesis. Functional evaluation revealed that mutant A and B subunits were stable and uncoupled from phosphatase activity. Mutant B56δ was A and C binding–deficient, while mutant Aα subunits bound B56δ well but were unable to bind C or bound a catalytically impaired C, suggesting a dominant-negative effect where mutant subunits hinder dephosphorylation of B56δ-anchored substrates. Moreover, mutant subunit overexpression resulted in hyperphosphorylation of GSK3β, a B56δ-regulated substrate. This effect was in line with clinical observations, supporting a correlation between the ID degree and biochemical disturbance. PMID:26168268

Constitutive Activation of the G-Protein Subunit G[alpha]s within Forebrain Neurons Causes PKA-Dependent Alterations in Fear Conditioning and Cortical "Arc" mRNA Expression

ERIC Educational Resources Information Center

Kelly, Michele P.; Cheung, York-Fong; Favilla, Christopher; Siegel, Steven J.; Kanes, Stephen J.; Houslay, Miles D.; Abel, Ted

2008-01-01

Memory formation requires cAMP signaling; thus, this cascade has been of great interest in the search for cognitive enhancers. Given that medications are administered long-term, we determined the effects of chronically increasing cAMP synthesis in the brain by expressing a constitutively active isoform of the G-protein subunit G[alpha]s…

Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

PubMed

Coopman, P J; Thomas, D M; Gehlsen, K R; Mueller, S C

1996-11-01

The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3 antibodies and the laminin peptide HGD-6 activate the alpha 3 beta 1 integrin, which results in a downstream signaling cascade stimulating phagocytosis.

Activation and inhibition of mouse muscle and neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

PubMed

Papke, Roger L; Wecker, Lynn; Stitzel, Jerry A

2010-05-01

Transgenic mouse models with nicotinic acetylcholine receptor (nAChR) knockouts and knockins have provided important insights into the molecular substrates of addiction and disease. However, most studies of heterologously expressed neuronal nAChR have used clones obtained from other species, usually human or rat. In this work, we use mouse clones expressed in Xenopus oocytes to provide a relatively comprehensive characterization of the three primary classes of nAChR: muscle-type receptors, heteromeric neuronal receptors, and homomeric alpha7-type receptors. We evaluated the activation of these receptor subtypes with acetylcholine and cytisine-related compounds, including varenicline. We also characterized the activity of classic nAChR antagonists, confirming the utility of mecamylamine and dihydro-beta-erythroidine as selective antagonists in mouse models of alpha3beta4 and alpha4beta2 receptors, respectively. We also conducted an in-depth analysis of decamethonium and hexamethonium on muscle and neuronal receptor subtypes. Our data indicate that, as with receptors cloned from other species, pairwise expression of neuronal alpha and beta subunits in oocytes generates heterogeneous populations of receptors, most likely caused by variations in subunit stoichiometry. Coexpression of the mouse alpha5 subunit had varying effects, depending on the other subunits expressed. The properties of cytisine-related compounds are similar for mouse, rat, and human nAChR, except that varenicline produced greater residual inhibition of mouse alpha4beta2 receptors than with human receptors. We confirm that decamethonium is a partial agonist, selective for muscle-type receptors, but also note that it is a nondepolarizing antagonist for neuronal-type receptors. Hexamethonium was a relatively nonselective antagonist with mixed competitive and noncompetitive activity.

UBXD4, a UBX-containing protein, regulates the cell surface number and stability of alpha3-containing nicotinic acetylcholine receptors.

PubMed

Rezvani, Khosrow; Teng, Yanfen; Pan, Yaping; Dani, John A; Lindstrom, Jon; García Gras, Eduardo A; McIntosh, J Michael; De Biasi, Mariella

2009-05-27

Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the alpha3 and alpha4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with alpha3-containing nAChRs (alpha3* nAChRs) expressed in HEK293 cells, PC12 cells, and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the alpha3beta2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining, and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of alpha3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the alpha3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of alpha3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the alpha3 subunit and, consequently, the number of receptors at the cell surface.

Molecular cloning of pituitary glycoprotein alpha-subunit and follicle stimulating hormone and chorionic gonadotropin beta-subunits from New World squirrel monkey and owl monkey.

PubMed

Scammell, Jonathan G; Funkhouser, Jane D; Moyer, Felricia S; Gibson, Susan V; Willis, Donna L

2008-02-01

The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone alpha-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature beta subunits of follicle stimulating hormone (FSHbeta) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHbeta. New World primate glycoprotein hormone alpha-polypeptides and FSHbeta subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the beta-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the beta-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGbeta are 143 and 144 amino acids in length and 77% homologous with human CGbeta. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGbeta, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGbetas. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.

ADP binding to TF1 and its subunits induces ultraviolet spectral changes.

PubMed

Hisabori, T; Yoshida, M; Sakurai, H

1986-09-01

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.

Subunit Compositions of the RNA-Silencing Enzymes Pol IV and Pol V Reveal Their Origins as Specialized Forms of RNA Polymerase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ream, Thomas S.; Haag, J. R.; Wierzbicki, A. T.

2009-01-30

In addition to RNA polymerases I, II and III, which are multi-subunit RNA polymerases found in all eukaryotes, plants have catalytic subunits for two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V (formerly Pol IVa and Pol IVb, respectively). Pol IV and Pol V play non-redundant roles in siRNA-directed DNA methylation and gene silencing pathways.

Direct evidence that FK506 inhibition of FcepsilonRI-mediated exocytosis from RBL mast cells involves calcineurin.

PubMed

Hultsch, T; Brand, P; Lohmann, S; Saloga, J; Kincaid, R L; Knop, J

1998-05-01

FcepsilonRI-mediated exocytosis of preformed mediators from mast cells and basophils (e.g. histamine, serotonin, beta-hexosaminidase) is sensitive to the immunosuppressants cyclosporin A and FK506 (IC50 200 and 4 nM, respectively) but not rapamycin. The mechanism of inhibition does not appear to involve tyrosine phosphorylation, hydrolysis of inositol phosphates or calcium flux. Here we report experiments using a molecular approach to assess the role of calcineurin, a serine/threonine phosphatase thought to be the primary pharmacological target of these drugs. Calcineurin's activity requires association of its catalytic (A) subunit with an intrinsic regulatory (B) subunit. We hypothesized that calcineurin-sensitive signalling events should be affected by the depletion of calcineurin B subunits, thereby reducing the number of active A:B complexes. We therefore transfected rat basophilic leukemia (RBL) cells with an inhibitory (dominant negative) form of the calcineurin A subunit, which binds the calcineurin B subunit with high affinity but does not possess catalytic activity (B subunit knock-out, BKO). In these transfected cells, the dose-response curve for the inhibition of FcepsilonRI-mediated exocytosis by FK506 was shifted to the left, indicating an increased drug sensitivity of BKO-transfected cells. We conclude that FK506 inhibition of FcepsilonRI-mediated exocytosis in mast cells specifically targets calcineurin activity.

Assembly of Q{beta} viral RNA polymerase with host translational elongation factors EF-Tu and -Ts.

PubMed

Takeshita, Daijiro; Tomita, Kozo

2010-09-07

Replication and transcription of viral RNA genomes rely on host-donated proteins. Qbeta virus infects Escherichia coli and replicates and transcribes its own genomic RNA by Qbeta replicase. Qbeta replicase requires the virus-encoded RNA-dependent RNA polymerase (beta-subunit), and the host-donated translational elongation factors EF-Tu and -Ts, as active core subunits for its RNA polymerization activity. Here, we present the crystal structure of the core Qbeta replicase, comprising the beta-subunit, EF-Tu and -Ts. The beta-subunit has a right-handed structure, and the EF-Tu:Ts binary complex maintains the structure of the catalytic core crevasse of the beta-subunit through hydrophobic interactions, between the finger and thumb domains of the beta-subunit and domain-2 of EF-Tu and the coiled-coil motif of EF-Ts, respectively. These hydrophobic interactions are required for the expression and assembly of the Qbeta replicase complex. Thus, EF-Tu and -Ts have chaperone-like functions in the maintenance of the structure of the active Qbeta replicase. Modeling of the template RNA and the growing RNA in the catalytic site of the Qbeta replicase structure also suggests that structural changes of the RNAs and EF-Tu:Ts should accompany processive RNA polymerization and that EF-Tu:Ts in the Qbeta replicase could function to modulate the RNA folding and structure.

Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

PubMed

Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

2014-08-15

The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

In vitro reconstitution of the active T. castaneum telomerase.

PubMed

Schuller, Anthony P; Harkisheimer, Michael J; Skordalakes, Emmanuel

2011-07-14

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro. Telomerase is a specialized reverse transcriptase that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit. Additionally, telomerase is dormant in most somatic cells in adults, but is active in cancer cells where it promotes cell immortality. The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres. Prior to 2008, only structures for individual telomerase domains had been solved. A major breakthrough in this field came from the determination of the crystal structure of the active, catalytic subunit of T. castaneum telomerase, TcTERT. Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

Alpha1B-adrenoceptor signaling and cell motility: GTPase function of Gh/transglutaminase 2 inhibits cell migration through interaction with cytoplasmic tail of integrin alpha subunits.

PubMed

Kang, Sung Koo; Yi, Kye Sook; Kwon, Nyoun Soo; Park, Kwang-Hyun; Kim, Uh-Hyun; Baek, Kwang Jin; Im, Mie-Jae

2004-08-27

A multifunctional enzyme, G(h), is a GTP-binding protein that couples to the alpha(1B)-adrenoreceptor and stimulates phospholipase C-delta1 but also displays transglutaminase 2 (TG2) activity. G(h)/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of G(h)/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the alpha(1)-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of G(h)/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the alpha(1B)-adrenoreceptor with the alpha(1) agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. G(h)/TG2 is coimmunoprecipitated by an integrin alpha(5) antibody and binds to the cytoplasmic tail peptide of integrins alpha(5), alpha(v), and alpha(IIb) subunits in the presence of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). Mutation of Lys-Arg residues in the GFFKR motif, present in the alpha(5)-tail, significantly reduces the binding of GTPgammaS-G(h)/TG2. Moreover, the motif-containing integrin alpha(5)-tail peptides block G(h)/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression G(h)/TG2. These results provide evidence that G(h) function initiates the modulation of cell motility via association of GTP-bound G(h)/TG2 with the GFFKR motif located in integrin alpha subunits.

Cardiac metabolic pathways affected in the mouse model of barth syndrome.

PubMed

Huang, Yan; Powers, Corey; Madala, Satish K; Greis, Kenneth D; Haffey, Wendy D; Towbin, Jeffrey A; Purevjav, Enkhsaikhan; Javadov, Sabzali; Strauss, Arnold W; Khuchua, Zaza

2015-01-01

Cardiolipin (CL) is a mitochondrial phospholipid essential for electron transport chain (ETC) integrity. CL-deficiency in humans is caused by mutations in the tafazzin (Taz) gene and results in a multisystem pediatric disorder, Barth syndrome (BTHS). It has been reported that tafazzin deficiency destabilizes mitochondrial respiratory chain complexes and affects supercomplex assembly. The aim of this study was to investigate the impact of Taz-knockdown on the mitochondrial proteomic landscape and metabolic processes, such as stability of respiratory chain supercomplexes and their interactions with fatty acid oxidation enzymes in cardiac muscle. Proteomic analysis demonstrated reduction of several polypeptides of the mitochondrial respiratory chain, including Rieske and cytochrome c1 subunits of complex III, NADH dehydrogenase alpha subunit 5 of complex I and the catalytic core-forming subunit of F0F1-ATP synthase. Taz gene knockdown resulted in upregulation of enzymes of folate and amino acid metabolic pathways in heart mitochondria, demonstrating that Taz-deficiency causes substantive metabolic remodeling in cardiac muscle. Mitochondrial respiratory chain supercomplexes are destabilized in CL-depleted mitochondria from Taz knockdown hearts resulting in disruption of the interactions between ETC and the fatty acid oxidation enzymes, very long-chain acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase, potentially affecting the metabolic channeling of reducing equivalents between these two metabolic pathways. Mitochondria-bound myoglobin was significantly reduced in Taz-knockdown hearts, potentially disrupting intracellular oxygen delivery to the oxidative phosphorylation system. Our results identify the critical pathways affected by the Taz-deficiency in mitochondria and establish a future framework for development of therapeutic options for BTHS.

Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit.

PubMed

Mellor, J R; Wisden, W; Randall, A D

2000-07-10

Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden, W., 1997. Ligand-gated ion channel partnerships: GABA(A) receptor alpha(6) subunit inactivation inhibits delta subunit expression. Journal of Neuroscience 17, 1350-1362).

Differentiation to adipocytes in accompanied by an increase in the amounts of Gi- and Go-proteins in 3T3-L1 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watkins, D.C.; Northup, J.K.; Malbon, C.C.

Treatment of cultures of 3T3-L1 cells with methylisobutyl-xanthine and dexamethasone has been shown to result in accumulation of lipid and conversion to the morphology of adipocytes in more than 90% of the cells. The status of the stimulatory (Gs), inhibitory (Gi) and Go-proteins during the course of 3T3-L1 differentiation was examined. The amount of alpha subunit of Gs (..cap alpha..Gs), assayed by radiolabeling in the presence of cholera toxin and (/sup 32/P)NAD/sup +/, increased upon differentiation as previously described by others. The amounts of ..cap alpha..Gi and ..cap alpha..Go assayed by radiolabeling in the presence of pertussis toxin and (/supmore » 32/P)NAD/sup +/ increased 3-fold upon differentiation. Immunoblots of cell membranes subjected to gel electrophoresis in sodium dodecyl sulfate were probed with two rabbit antisera raised against bovine brain ..cap alpha..Go and with one raised against the..beta..-subunit of the bovine rod-outer-segment G-protein, referred to as transducin. The immunoblotting data confirm the increase upon differentiation of ..cap alpha..Go and also demonstrate an increase in the amount of the ..beta..-subunit. Thus differentiation of 3T3-L1 cells is accompanied by dramatic changes in the complexion of G-proteins in the membranes.« less

Expression of {beta}{sub 1} integrins in human endometrial stromal and decidual cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shiokawa, Shigetatsu; Yoshimura, Yasunori; Nakamura, Yukio

The present study was undertaken to investigate the expression of {beta}{sub 1} integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, and {alpha}{sub 5} subunits of the {beta}{sub 1} integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E{sub 2}) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of {beta}{sub 1} integrins in vitro. Flow cytometry also demonstrated the expression of themore » {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, {alpha}{sub 3}, {alpha}{sub 5}, and {alpha}{sub 6} subunits of {beta}{sub 1} integrin family in cultured decidual cells, and the enriched-fraction of prolactin (PRL)-producing decidual cells isolated by Percoll gradients showed high levels of {beta}{sub 1} integrins expression. Immunohistochemistry confirmed the {beta}{sub 1} integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. In summary, the present study demonstrated that endometrial stromal and decidual cells expressed {beta}{sub 1} integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, {beta}{sub 1} integrins in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation. 43 refs., 7 figs., 2 tabs.« less

Mutant POLG2 Disrupts DNA Polymerase γ Subunits and Causes Progressive External Ophthalmoplegia

PubMed Central

Longley, Matthew J.; Clark, Susanna; Yu Wai Man, Cynthia; Hudson, Gavin; Durham, Steve E.; Taylor, Robert W.; Nightingale, Simon; Turnbull, Douglass M.; Copeland, William C.; Chinnery, Patrick F.

2006-01-01

DNA polymerase γ (pol γ) is required to maintain the genetic integrity of the 16,569-bp human mitochondrial genome (mtDNA). Mutation of the nuclear gene for the catalytic subunit of pol γ (POLG) has been linked to a wide range of mitochondrial diseases involving mutation, deletion, and depletion of mtDNA. We describe a heterozygous dominant mutation (c.1352G→A/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol γ, that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)–deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype. PMID:16685652

The prokaryote-to-eukaryote transition reflected in the evolution of the V/F/A-ATPase catalytic and proteolipid subunits

NASA Technical Reports Server (NTRS)

Hilario, E.; Gogarten, J. P.

1998-01-01

Changes in the primary and quarternary structure of vacuolar and archaeal type ATPases that accompany the prokaryote-to-eukaryote transition are analyzed. The gene encoding the vacuolar-type proteolipid of the V-ATPase from Giardia lamblia is reported. Giardia has a typical vacuolar ATPase as observed from the common motifs shared between its proteolipid subunit and other eukaryotic vacuolar ATPases, suggesting that the former enzyme works as a hydrolase in this primitive eukaryote. The phylogenetic analyses of the V-ATPase catalytic subunit and the front and back halves of the proteolipid subunit placed Giardia as the deepest branch within the eukaryotes. Our phylogenetic analysis indicated that at least two independent duplication and fusion events gave rise to the larger proteolipid type found in eukaryotes and in Methanococcus. The spatial distribution of the conserved residues among the vacuolar-type proteolipids suggest a zipper-type interaction among the transmembrane helices and surrounding subunits of the V-ATPase complex. Important residues involved in the function of the F-ATP synthase proteolipid have been replaced during evolution in the V-proteolipid, but in some cases retained in the archaeal A-ATPase. Their possible implication in the evolution of V/F/A-ATPases is discussed.

Differences in cholinergic responses from outer hair cells of rat and guinea pig.

PubMed

Chen, C; LeBlanc, C; Bobbin, R P

1996-09-01

A cholinergic receptor on outer hair cells (OHC) in guinea pig cochlea induces a K+ current when it is activated by acetylcholine and suberyldicholine but not by nicotine or muscarine (Bobbin, 1995). This unusual receptor may contain an alpha 9-subunit. However, the pharmacology of the alpha 9-subunit cloned from rat and expressed in Xenopus oocytes does not completely match that obtained for the ACh receptor in guinea pig OHCs. The response to 1,1-dimethyl-4-phenylpiperazinium (DMPP) is large in guinea pig OHCs and small in oocytes containing receptors of the alpha 9-subunit. Therefore, we compared the effects of cholinergic receptor agonists in rat and guinea pig OHCs using the whole-cell variant of the patch-clamp technique. ACh caused the largest outward K+ current in OHCs from both rat and guinea pig. Carbachol- and suberyldicholine-induced responses were similar in magnitude in OHCs of rat and guinea pig. However, DMPP produced a small response in OHCs from rat and a large response in OHCs from guinea pig. At a concentration of 100 microM, muscarine, oxotremorine M, nicotine and cytisine induced little response in guinea pig OHCs and none in rat OHCs. Results suggest that the ACh receptor on rat OHCs is similar to the alpha 9-subunit-containing receptor expressed in oocytes but different from the ACh receptor on guinea pig OHCs.

Identification of the electron transfer flavoprotein as an upregulated enzyme in the benzoate utilization of Desulfotignum balticum.

PubMed

Habe, Hiroshi; Kobuna, Akinori; Hosoda, Akifumi; Kosaka, Tomoyuki; Endoh, Takayuki; Tamura, Hiroto; Yamane, Hisakazu; Nojiri, Hideaki; Omori, Toshio; Watanabe, Kazuya

2009-07-01

Desulfotignum balticum utilizes benzoate coupled to sulfate reduction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis was conducted to detect proteins that increased more after growth on benzoate than on butyrate. A comparison of proteins on 2D gels showed that at least six proteins were expressed. The N-terminal sequences of three proteins exhibited significant identities with the alpha and beta subunits of electron transfer flavoprotein (ETF) from anaerobic aromatic-degraders. By sequence analysis of the fosmid clone insert (37,590 bp) containing the genes encoding the ETF subunits, we identified three genes, whose deduced amino acid sequences showed 58%, 74%, and 62% identity with those of Gmet_2267 (Fe-S oxidoreductase), Gmet_2266 (ETF beta subunit), and Gmet_2265 (ETF alpha subunit) respectively, which exist within the 300-kb genomic island of aromatic-degradation genes from Geobacter metallireducens GS-15. The genes encoding ETF subunits found in this study were upregulated in benzoate utilization.

Lewis base activation of Lewis acids: catalytic, enantioselective addition of glycolate-derived silyl ketene acetals to aldehydes.

PubMed

Denmark, Scott E; Chung, Won-Jin

2008-06-20

A catalytic system involving silicon tetrachloride and a chiral, Lewis basic bisphosphoramide catalyst is effective for the addition of glycolate-derived silyl ketene acetals to aldehydes. It was found that the sense of diastereoselectivity could be modulated by changing the size of the substituents on the silyl ketene acetals. In general, the trimethylsilyl ketene acetals derived from methyl glycolates with a large protecting group on the alpha-oxygen provide enantiomerically enriched alpha,beta-dihydroxy esters with high syn-diastereoselectivity, whereas the tert-butyldimethylsilyl ketene acetals derived from bulky esters of alpha-methoxyacetic acid provide enantiomerically enriched alpha,beta-dihydroxy esters with high anti-diastereoselecitvity.

Nucleotide-Protectable Labeling of Sulfhydryl Groups in Subunit I of the ATPhase from Halobacterium Saccharovorum

NASA Technical Reports Server (NTRS)

Sulzner, Michael; Stan-Lotter, Helga; Hochstein, Lawrence I.

1992-01-01

A membrane-bound ATPase from the archaebacterium Halobacterium saccharovorum is inhibited by N-ethyl-maleimide in a nucleotide-protectable manner. When the enzyme was incubated with N-[C-14]jethylmaleimide, the bulk of radioactivity was as- sociated with the 87,000-Da subunit (subunit 1). ATP, ADP, or AMP reduced incorporation of the inhibitor. No charge shift of subunit I was detected following labeling with N-ethylmaleimide, indicating an electroneutral reaction. The results are consistent with the selective modification of sulfhydryl groups in subunit I at or near the catalytic site and are further evidence of a resemblance between this archaebacterial ATPase and the vacuolar-type ATPases.

Binding Linkage in a Telomere DNA–Protein Complex at the Ends of Oxytricha nova Chromosomes

PubMed Central

Buczek, Pawel; Orr, Rochelle S.; Pyper, Sean R.; Shum, Mili; Ota, Emily Kimmel Irene; Gerum, Shawn E.; Horvath, Martin P.

2005-01-01

Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein–protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (KD-DNA=1.4 nM). Another fusion protein, constructed without the C-terminal protein–protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (KD-DNA=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA–protein stability to protein–protein contacts at a remote site may provide a trigger point for DNA–protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase. PMID:15967465

Mutations in the thyrotropin receptor signal transduction pathway in the hyperfunctioning thyroid nodules from multinodular goiters: a study in the Turkish population.

PubMed

Gozu, Hulya; Avsar, Melike; Bircan, Rifat; Sahin, Serap; Deyneli, Oguzhan; Cirakoglu, Beyazit; Akalin, Sema

2005-10-01

Many studies have been carried out to determine G(s) alpha and TSHR mutations in autonomously functioning thyroid nodules. Variable prevalences for somatic constitutively activating TSHR mutations in hot nodules have been reported. Moreover, the increased prevalence of toxic multinodular goiters in iodine-deficient regions is well known. In Turkey, a country with high incidence rates of goiter due to iodine deficiency, the frequency of mutations in the thyrotropin receptor signal transduction pathway has not been evaluated up to now. In the present study, a part of the genes of the TSHR, G(s)alpha and the catalytic subunit of the PKA were checked for activating mutations. Thirty-five patients who underwent thyroidectomy for multinodular goiters were examined. Genomic DNAs were extracted from 58 hyperactive nodular specimens and surrounding normal thyroid tissues. Mutation screening was done by single-strand conformational polymorphism (SSCP) analysis. In those cases where a mutation was detected, the localization of the mutation was determined by automatic DNA sequencing. No G(s)alpha or PKA mutations were detected, whereas ten mutations (17%) were identified in the TSHR gene. All mutations were somatic and heterozygotic. In conclusion, the frequency of mutations in the cAMP signal transduction pathway was found to be lower than expected in the Turkish population most likely because of the use of SSCP as a screening method and sequencing only a part of TSHR exon 10.

Neuronal nicotinic acetylcholine receptor subunits in autism: an immunohistochemical investigation in the thalamus.

PubMed

Ray, M A; Graham, A J; Lee, M; Perry, R H; Court, J A; Perry, E K

2005-08-01

The cholinergic system has been implicated in the development of autism on the basis of neuronal nicotinic acetylcholine receptor (nAChR) losses in cerebral and cerebellar cortex. In the present study, the first to explore nAChRs in the thalamus in autism, alpha4, alpha7 and beta2 nAChR subunit expression in thalamic nuclei of adult individuals with autism (n=3) and age-matched control cases (n=3) was investigated using immunochemical methods. Loss of alpha7- and beta2- (but not alpha4-) immunoreactive neurons occurred in the paraventricular nucleus (PV) and nucleus reuniens in autism. Preliminary results indicated glutamic acid decarboxylase immunoreactivity occurred at a low level in PV, co-expressed with alpha7 in normal and autistic cases and was not reduced in autism. This suggested loss of neuronal alpha7 in autism is not caused by loss of GABAergic neurons. These findings indicate nicotinic abnormalities that occur in the thalamus in autism which may contribute to sensory or attentional deficits.

Arginine 26 and aspartic acid 69 of the regulatory subunit are key residues of subunits interaction of acetohydroxyacid synthase isozyme III from E. coli.

PubMed

Zhao, Yuefang; Wen, Xin; Niu, Congwei; Xi, Zhen

2012-11-05

Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is composed of catalytic and regulatory subunits. The enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). However, the crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and RSU is also unknown. Herein, we introduced a global-surface, site-directed labeling scanning method to determine the potential interaction region of the RSU. This approach relies on the insertion of a bulky fluorescent probe at the designated site on the surface of the RSU to cause a dramatic change in holoenzyme activity by perturbing subunit interaction. Then, the key amino acid residues in the potential interaction regions were identified by site-directed mutagenesis. Compared to the wild-type, the single-point mutants R26A and D69A showed 54 and 64 % activity, respectively, whereas the double mutant (R26A+D69A) gave 14 %, thus suggesting that residues Arg26 and Asp69 are the key residues of subunit interaction with cooperative action. Additionally, the results of GST pull-down assays and pH-dependence experiments suggested that polar interaction is the main force for subunits interaction. A plausible protein-protein interaction model of the holoenzyme of Escherichia coli AHAS III is proposed, based on the mutagenesis and protein docking studies. The protocol established here should be useful for the identification of the molecular interactions between proteins. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

DOE Office of Scientific and Technical Information (OSTI.GOV)

Middleton, R.E.; Cohen, J.B.

1991-07-16

The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with highmore » affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.« less

Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv.

PubMed

Kaydamov, C; Tewes, A; Adler, K; Manteuffel, R

2000-04-25

We have isolated cDNA sequences encoding alpha and beta subunits of potential G proteins from a cDNA library prepared from somatic embryos of Nicotiana plumbaginifolia Viv. at early developmental stages. The predicted NPGPA1 and NPGPB1 gene products are 75-98% identical to the known respective plant alpha and beta subunits. Southern hybridizations indicate that NPGPA1 is probably a single-copy gene, whereas at least two copies of NPGPB1 exist in the N. plumbaginifolia genome. Northern analyses reveal that both NPGPA1 and NPGPB1 mRNA are expressed in all embryogenic stages and plant tissues examined and their expression is obviously regulated by the plant hormone auxin. Immunohistological localization of NPGPalpha1 and NPGPbeta1 preferentially on plasma and endoplasmic reticulum membranes and their immunochemical detection exclusively in microsomal cell fractions implicate membrane association of both proteins. The temporal and spatial expression patterns of NPGPA1 and NPGPB1 show conformity as well as differences. This could account for not only cooperative, but also individual activities of both subunits during embryogenesis and plant development.

Hyperthyroidism secondary to a pituitary adenoma secreting TSH, FSH, alpha-subunit and GH.

PubMed

Patrick, A W; Atkin, S L; MacKenzie, J; Foy, P M; White, M C; MacFarlane, I A

1994-02-01

A 51-year-old man had been treated for hyperthyroidism with antithyroid drugs for 8 years. He was then found to have a large pituitary adenoma with biochemical evidence of overproduction of TSH, FSH and alpha-subunit. Subsequent immunocytochemical and tissue culture studies confirmed secretion of these hormones. In addition, the tumour stained for GH and was capable of GH production in vitro. This combination of hormones produced by a pituitary adenoma has not been previously reported.

MtGimC, a novel archaeal chaperone related to the eukaryotic chaperonin cofactor GimC/prefoldin.

PubMed

Leroux, M R; Fändrich, M; Klunker, D; Siegers, K; Lupas, A N; Brown, J R; Schiebel, E; Dobson, C M; Hartl, F U

1999-12-01

Group II chaperonins in the eukaryotic and archaeal cytosol assist in protein folding independently of the GroES-like cofactors of eubacterial group I chaperonins. Recently, the eukaryotic chaperonin was shown to cooperate with the hetero-oligomeric protein complex GimC (prefoldin) in folding actin and tubulins. Here we report the characterization of the first archaeal homologue of GimC, from Methanobacterium thermoautotrophicum. MtGimC is a hexamer of 87 kDa, consisting of two alpha and four beta subunits of high alpha-helical content that are predicted to contain extended coiled coils and represent two evolutionarily conserved classes of Gim subunits. Reconstitution experiments with MtGimC suggest that two subunits of the alpha class (archaeal Gimalpha and eukaryotic Gim2 and 5) form a dimer onto which four subunits of the beta class (archaeal Gimbeta and eukaryotic Gim1, 3, 4 and 6) assemble. MtGimalpha and beta can form hetero-complexes with yeast Gim subunits and MtGimbeta partially complements yeast strains lacking Gim1 and 4. MtGimC is a molecular chaperone capable of stabilizing a range of non-native proteins and releasing them for subsequent chaperonin-assisted folding. In light of the absence of Hsp70 chaperones in many archaea, GimC may fulfil an ATP-independent, Hsp70-like function in archaeal de novo protein folding.

MtGimC, a novel archaeal chaperone related to the eukaryotic chaperonin cofactor GimC/prefoldin.

PubMed Central

Leroux, M R; Fändrich, M; Klunker, D; Siegers, K; Lupas, A N; Brown, J R; Schiebel, E; Dobson, C M; Hartl, F U

1999-01-01

Group II chaperonins in the eukaryotic and archaeal cytosol assist in protein folding independently of the GroES-like cofactors of eubacterial group I chaperonins. Recently, the eukaryotic chaperonin was shown to cooperate with the hetero-oligomeric protein complex GimC (prefoldin) in folding actin and tubulins. Here we report the characterization of the first archaeal homologue of GimC, from Methanobacterium thermoautotrophicum. MtGimC is a hexamer of 87 kDa, consisting of two alpha and four beta subunits of high alpha-helical content that are predicted to contain extended coiled coils and represent two evolutionarily conserved classes of Gim subunits. Reconstitution experiments with MtGimC suggest that two subunits of the alpha class (archaeal Gimalpha and eukaryotic Gim2 and 5) form a dimer onto which four subunits of the beta class (archaeal Gimbeta and eukaryotic Gim1, 3, 4 and 6) assemble. MtGimalpha and beta can form hetero-complexes with yeast Gim subunits and MtGimbeta partially complements yeast strains lacking Gim1 and 4. MtGimC is a molecular chaperone capable of stabilizing a range of non-native proteins and releasing them for subsequent chaperonin-assisted folding. In light of the absence of Hsp70 chaperones in many archaea, GimC may fulfil an ATP-independent, Hsp70-like function in archaeal de novo protein folding. PMID:10581246

Crystal Structure of Human [Beta]-Hexosaminidase B: Understanding the Molecular Basis of Sandhoff and Tay-Sachs Disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mark, Brian L.; Mahuran, Don J.; Cherney, Maia M.

2010-12-01

In humans, two major {beta}-hexosaminidase isoenzymes exist: Hex A and Hex B. Hex A is a heterodimer of subunits {alpha} and {beta} (60% identity), whereas Hex B is a homodimer of {beta}-subunits. Interest in human {beta}-hexosaminidase stems from its association with Tay-Sachs and Sandhoff disease; these are prototypical lysosomal storage disorders resulting from the abnormal accumulation of G{sub M2}-ganglioside (G{sub M2}). Hex A degrades G{sub M2} by removing a terminal N-acetyl-D-galactosamine ({beta}-GalNAc) residue, and this activity requires the G{sub M2}-activator, a protein which solubilizes the ganglioside for presentation to Hex A. We present here the crystal structure of human Hexmore » B, alone (2.4 {angstrom}) and in complex with the mechanistic inhibitors GalNAc-isofagomine (2.2 {angstrom}) or NAG-thiazoline (2.5 {angstrom}). From these, and the known X-ray structure of the G{sub M2}-activator, we have modeled Hex A in complex with the activator and ganglioside. Together, our crystallographic and modeling data demonstrate how {alpha} and {beta}-subunits dimerize to form either Hex A or Hex B, how these isoenzymes hydrolyze diverse substrates, and how many documented point mutations cause Sandhoff disease ({beta}-subunit mutations) and Tay-Sachs disease ({alpha}-subunit mutations).« less

O-linked oligosaccharides on insulin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collier, E.; Gorden, P.

1991-02-01

The insulin receptor, an integral membrane glycoprotein, is synthesized as a single-chain precursor that is cleaved to produce two mature subunits, both of which contain N-linked oligosaccharide chains and covalently linked fatty acids. We report that the beta-subunit also contains O-linked oligosaccharides. The proreceptor, alpha-subunit, and beta-subunit were labeled with (3H)mannose and (3H)galactose in the presence or absence of an inhibitor of O-linked glycosylation. Tryptic peptides from each component were separated by reverse-phase high-performance liquid chromatography. N- and O-linked oligosaccharide chains were identified on these peptides by specific enzymatic digestions. The proreceptor and alpha-subunit contained only N-linked oligosaccharides, whereas themore » beta-subunit contained both N- and O-linked oligosaccharides. The O-linked oligosaccharide chains were attached to a single tryptic fraction of the beta-subunit, which also contained N-linked chains. This fraction was further localized to the NH2-terminal tryptic peptide of the beta-subunit by specific immunoprecipitation with an anti-peptide antibody with specificity for this region. Binding of insulin and autophosphorylation of the beta-subunit were not dependent on O-linked glycosylation, because cells grown in the presence of the inhibitor exhibited a normal dose response to insulin. Therefore, the insulin receptor contains O-linked oligosaccharides on the NH2-terminal tryptic peptide of the beta-subunit, and these O-linked oligosaccharides are not necessary to the binding or autophosphorylation function of the receptor.« less

Complex formation between the protein components of methane monooxygenase from Methylosinus trichosporium OB3b. Identification of sites of component interaction.

PubMed

Fox, B G; Liu, Y; Dege, J E; Lipscomb, J D

1991-01-05

Kinetic, spectroscopic, and chemical evidence for the formation of specific catalytically essential complexes between the three protein components of the soluble form of methane monooxygenase from Methylosinus trichosporium OB3b is reported. The effects of the concentrations of the reductase and component B on the hydroxylation activity of the reconstituted enzyme system has been numerically simulated based on a kinetic model which assumes formation of multiple high affinity complexes with the hydroxylase component during catalysis. The formation of several of these complexes has been directly demonstrated. By using EPR spectroscopy, the binding of approximately 2 mol of component B/mol of hydroxylase (subunit structure (alpha beta gamma)2) is shown to significantly change the electronic environment of the mu-(H/R)-oxo-bridged binuclear iron cluster of the hydroxylase in both the mixed valent (Fe(II).Fe(III)) and fully reduced (Fe(II).Fe(II)) states. Protein-protein complexes between the reductase and component B as well as between the reductase and hydroxylase have been shown to form by monitoring quenching of the tryptophan fluorescence spectrum of either the component B (KD approximately 0.4 microM) or hydroxylase (two binding sites, KDa approximately 10 nM, KDb approximately 8 microM). The observed KD values are in agreement with the best fit values from the kinetic simulation. Through the use of the covalent zero length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), the binding sites of the component B and reductase were shown to be on the hydroxylase alpha and beta subunits, respectively. The alpha and beta subunits of the hydroxylase are cross-linked by EDC suggesting that they are juxtaposed. EDC also caused the rapid loss of the ability of the monomeric component B to stimulate the hydroxylation reaction suggesting that cross-linking of reactive groups on the protein surface had occurred. This effect was inhibited by the presence of hydroxylase and was accompanied by a loss of the ability of the component B to bind to the hydroxylase. Thus, formation of a component B-hydroxylase complex is apparently required for effective catalysis linked to NADH oxidation. When present in concentrations greater than required to saturate the initial hydroxylase complex, component B inhibited both the rate of the enzymic reaction and the cross-linking of the reductase to the hydroxylase. This suggests that a second complex involving component B can form that negatively regulates catalysis by preventing formation of the reductase-hydroxylase complex.

Regulation of protein phosphatase 2A during embryonic diapause process in the silkworm, Bombyx mori.

PubMed

Gu, Shi-Hong; Hsieh, Hsiao-Yen; Lin, Pei-Ling

2017-11-01

Regulation of protein phosphorylation requires coordinated interactions between protein kinases and protein phosphatases. In the present study, we investigated regulation of protein phosphatase 2A (PP2A) during the embryonic diapause process of B. mori. An immunoblotting analysis showed that Bombyx eggs contained a catalytic C subunit, a major regulatory B subunit (B55/PR55 subunit), and a structural A subunit, with the A and B subunits undergoing differential changes between diapause and non-diapause eggs during embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5°C for 70days and then were transferred to 25°C, protein levels of the A and B subunits of PP2A gradually increased toward embryonic development. However, protein levels of the A and B subunits in diapause eggs remained at low levels during the first 8days after oviposition. The direct determination of PP2A enzymatic activity showed that the activity remained at low levels in diapause eggs during the first 8days after oviposition. However, in non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling, PP2A enzymatic activity sharply increased during the first several days, reached a peak during the middle embryonic development, and then greatly decreased 3 or 4days before hatching. Examination of temporal changes in mRNA expression levels of the catalytic β subunit and regulatory subunit of PP2A showed high levels in eggs whose diapause initiation was prevented by HCl compared to those in diapause eggs. These results demonstrate that the higher PP2A gene expression and PP2A A and B subunit protein levels and increased enzymatic activity are related to embryonic development of B. mori. Copyright © 2017 Elsevier Ltd. All rights reserved.

Affective and cognitive effects of global deletion of alpha3-containing gamma-aminobutyric acid-A receptors.

PubMed

Fiorelli, Roberto; Rudolph, Uwe; Straub, Carolin J; Feldon, Joram; Yee, Benjamin K

2008-09-01

Gamma-aminobutyric acid (GABA)A receptors characterized by the presence of the alpha3 subunit are the major GABAA receptor subtype expressed in brain stem monoaminergic nuclei. These alpha3-GABAA receptors are therefore in a unique position to regulate monoaminergic functions. To characterize the functional properties of alpha3-GABAA receptors, we present a preliminary assessment of the expression of affective and cognitive behaviour in male mice with a targeted deletion of the Gabra3 gene encoding the alpha3 subunit [alpha3 knockout (KO) mice] on a C57BL/6Jx129X1/SvJ F1 hybrid genetic background. The alpha3 KO mice did not exhibit any gross change of anxiety-like behaviour or spontaneous locomotor behaviour. In the Porsolt forced swim test for potential antidepressant activity, alpha3 KO mice exhibited reduced floating and enhanced swimming behaviour relative to wild-type controls. Performance on a two-choice sucrose preference test, however, revealed no evidence for an increase in sucrose preference in the alpha3 KO mice that would have substantiated a potential phenotype for depression-related behaviour. In contrast, a suggestion of an enhanced negative contrast effect was revealed in a one-bottle sucrose consumption test across different sucrose concentrations. These affective phenotypes were accompanied by alterations in the balance between conditioned responding to the discrete conditioned stimulus and to the context, and a suggestion of faster extinction, in the Pavlovian conditioned freezing paradigm. Spatial learning in the water maze reference memory test, however, was largely unchanged in the alpha3 KO mice, except for a trend of preservation during reversal learning. The novel phenotypes following global deletion of the GABAA receptor alpha3 subunit identified here provided relevant insights, in addition to our earlier study, into the potential behavioural relevance of this specific receptor subtypes in the modulation of both affective and cognitive functions.

Functional characterization of mongoose nicotinic acetylcholine receptor alpha-subunit: resistance to alpha-bungarotoxin and high sensitivity to acetylcholine.

PubMed

Asher, O; Lupu-Meiri, M; Jensen, B S; Paperna, T; Fuchs, S; Oron, Y

1998-07-24

The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) alpha-subunit contains a number of mutations in the ligand-binding domain and exhibits poor binding of alpha-bungarotoxin (alpha-BTX). We characterized the functional properties of a hybrid (alpha-mongoose/beta gamma delta-rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of alpha-BTX to the hybrid AChR was 200-fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 microM for the rat AChR). Hence, changes in the ligand-binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.

Human cytomegalovirus carries serine/threonine protein phosphatases PP1 and a host-cell derived PP2A.

PubMed Central

Michelson, S; Turowski, P; Picard, L; Goris, J; Landini, M P; Topilko, A; Hemmings, B; Bessia, C; Garcia, A; Virelizier, J L

1996-01-01

Human cytomegalovirus (CMV), a herpesvirus, is an important cause of morbidity and mortality in immunocompromised patients. When studying hyper-immediate-early events after contact between CMV virions and the cell membrane, we observed a hypophosphorylation of cellular proteins within 10 min. This can be explained in part by our finding that purified CMV contains serine/threonine protein phosphatase activities. Biochemical analyses indicate that this protein phosphatase activity has all characteristics of type 1 and 2A protein phosphatases (PP1 and PP2A). Specifically, PP1 accounts for approximately 30% and PP2A accounts for the remaining 70% of the phosphorylase phosphatase activity found. CMV produced in astrocytoma cells stably expressing an amino-terminally tagged PP2A catalytic subunit contained tagged enzyme, thus demonstrating the cellular origin of CMV-associated PP2A. PP2A is specifically found inside the virus, associated with the nucleocapsid fraction. Western blot (immunoblot) analysis of purified virus revealed the presence of the catalytic subunits of PP2A and PP1. Furthermore, the catalytic subunit of PP2A appears to be complexed to the regulatory subunits PR65 and PR55, which is also the most abundant configuration of this enzyme found in the host cells. Incubation of virus with okadaic acid before contact of CMV with cells prevented hypophosphorylation of cellular proteins, thus demonstrating the role of CMV-associated phosphatases in this phenomenon. CMV can thus transport an active enzyme from one cell to another. PMID:8627658

Structural Asymmetry and Disulfide Bridges among Subunits Modulate the Activity of Human Malonyl-CoA Decarboxylase*

PubMed Central

Aparicio, David; Pérez-Luque, Rosa; Carpena, Xavier; Díaz, Mireia; Ferrer, Joan C.; Loewen, Peter C.; Fita, Ignacio

2013-01-01

Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is an essential facet in the regulation of fatty acid metabolism. The structure of human peroxisomal MCD reveals a molecular tetramer that is best described as a dimer of structural heterodimers, in which the two subunits present markedly different conformations. This molecular organization is consistent with half-of-the-sites reactivity. Each subunit has an all-helix N-terminal domain and a catalytic C-terminal domain with an acetyltransferase fold (GNAT superfamily). Intersubunit disulfide bridges, Cys-206–Cys-206 and Cys-243–Cys-243, can link the four subunits of the tetramer, imparting positive cooperativity to the catalytic process. The combination of a half-of-the-sites mechanism within each structural heterodimer and positive cooperativity in the tetramer produces a complex regulatory picture that is further complicated by the multiple intracellular locations of the enzyme. Transport into the peroxisome has been investigated by docking human MCD onto the peroxisomal import protein peroxin 5, which revealed interactions that extend beyond the C-terminal targeting motif. PMID:23482565

Polymerase ε1 mutation in a human syndrome with facial dysmorphism, immunodeficiency, livedo, and short stature (“FILS syndrome”)

PubMed Central

Pachlopnik Schmid, Jana; Lemoine, Roxane; Nehme, Nadine; Cormier-Daire, Valéry; Revy, Patrick; Debeurme, Franck; Debré, Marianne; Nitschke, Patrick; Bole-Feysot, Christine; Legeai-Mallet, Laurence; Lim, Annick; de Villartay, Jean-Pierre; Picard, Capucine; Durandy, Anne; Fischer, Alain

2012-01-01

DNA polymerase ε (Polε) is a large, four-subunit polymerase that is conserved throughout the eukaryotes. Its primary function is to synthesize DNA at the leading strand during replication. It is also involved in a wide variety of fundamental cellular processes, including cell cycle progression and DNA repair/recombination. Here, we report that a homozygous single base pair substitution in POLE1 (polymerase ε 1), encoding the catalytic subunit of Polε, caused facial dysmorphism, immunodeficiency, livedo, and short stature (“FILS syndrome”) in a large, consanguineous family. The mutation resulted in alternative splicing in the conserved region of intron 34, which strongly decreased protein expression of Polε1 and also to a lesser extent the Polε2 subunit. We observed impairment in proliferation and G1- to S-phase progression in patients’ T lymphocytes. Polε1 depletion also impaired G1- to S-phase progression in B lymphocytes, chondrocytes, and osteoblasts. Our results evidence the developmental impact of a Polε catalytic subunit deficiency in humans and its causal relationship with a newly recognized, inherited disorder. PMID:23230001

Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu, Y.

1989-01-01

The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the {alpha}-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced {sup 22}Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the {gamma}, {delta}, and {epsilon} subunit, respectively,more » of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four ({alpha}, {beta}, {gamma}, and {delta}) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the {alpha}, {beta}, and {delta} subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the {gamma}- and {epsilon}- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the {gamma} is replaced by {epsilon}.« less

Human brain nicotinic receptors, their distribution and participation in neuropsychiatric disorders.

PubMed

Graham, A J; Martin-Ruiz, C M; Teaktong, T; Ray, M A; Court, J A

2002-08-01

Mapping of nicotinic acetylcholine receptor (nAChR) subtypes and subunits in human brain is far from complete, however it is clear that multiple subunits are present (including alpha3, alpha4, alpha5, alpha6 and alpha7, beta2, alpha3 and beta4) and that these receptors are not solely distributed on neurones, but also on cerebral vasculature and astrocytes. It is important to elucidate subunit composition of receptors associated with different cell types and pathways within the human CNS in terms of potential nicotinic therapy for a range of both developmental and age-related disorders in which nAChR attenuation occurs. Reductions in nAChRs are reported in Alzheimer's and Parkinson's diseases, dementia with Lewy bodies, schizophrenia and autism, but may not be associated with reduced cortical cholinergic innervation observed in vascular dementia or occur at an early stage in Down's syndrome. Changes in nAChR expression in neuropsychiatric disorders appear to be brain region and subtype specific and have been shown in some instances to be associated with pathology and symptomatology. It is likely that deficits in alpha4-containing receptors predominate in cortical areas in Alzheimer's disease and autism, whereas reduction of alpha7 receptors may be more important in schizophrenia. Changes in astrocytic and vascular nAChR expression in neurodegenerative diseases should also be considered. Studies using both animal models and human autopsy tissue suggest that nAChRs can play a role in neuroprotection against age-related pathology. It is possible that the development of nAChR subtype specific drugs may lead to advances in therapy for both age-related and psychiatric disorders.

Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria.

PubMed

Whitney, Spencer M; Sharwood, Robert E; Orr, Douglas; White, Sarah J; Alonso, Hernan; Galmés, Jeroni

2011-08-30

Improving global yields of important agricultural crops is a complex challenge. Enhancing yield and resource use by engineering improvements to photosynthetic carbon assimilation is one potential solution. During the last 40 million years C(4) photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO(2)-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C(3) ancestors, C(4) plants combine a faster rubisco with a biochemical CO(2)-concentrating mechanism, enabling more efficient use of water and nitrogen and enhanced yield. Here we show the versatility of plastome manipulation in tobacco for identifying sequences in C(4)-rubisco that can be transplanted into C(3)-rubisco to improve carboxylation rate (V(C)). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C(3)), Flaveria floridana (C(3)-C(4)), and Flaveria bidentis (C(4)), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C(4) ((309)Ile; faster V(C), lower CO(2) affinity) and C(3) ((309)Met; slower V(C), higher CO(2) affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco V(C) in C(3) crops. Coengineering a catalytically faster C(3) rubisco and a CO(2)-concentrating mechanism within C(3) crop species could enhance their efficiency in resource use and yield.

Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria

PubMed Central

Whitney, Spencer M.; Sharwood, Robert E.; Orr, Douglas; White, Sarah J.; Alonso, Hernan; Galmés, Jeroni

2011-01-01

Improving global yields of important agricultural crops is a complex challenge. Enhancing yield and resource use by engineering improvements to photosynthetic carbon assimilation is one potential solution. During the last 40 million years C4 photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C3 ancestors, C4 plants combine a faster rubisco with a biochemical CO2-concentrating mechanism, enabling more efficient use of water and nitrogen and enhanced yield. Here we show the versatility of plastome manipulation in tobacco for identifying sequences in C4-rubisco that can be transplanted into C3-rubisco to improve carboxylation rate (VC). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C3), Flaveria floridana (C3-C4), and Flaveria bidentis (C4), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C4 (309Ile; faster VC, lower CO2 affinity) and C3 (309Met; slower VC, higher CO2 affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco VC in C3 crops. Coengineering a catalytically faster C3 rubisco and a CO2-concentrating mechanism within C3 crop species could enhance their efficiency in resource use and yield. PMID:21849620

Inhibition of liver fibrosis by solubilized coenzyme Q10: Role of Nrf2 activation in inhibiting transforming growth factor-beta1 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Hoo-Kyun; Pokharel, Yuba Raj; Lim, Sung Chul

2009-11-01

Coenzyme Q10 (CoQ10), an endogenous antioxidant, is important in oxidative phosphorylation in mitochondria. It has anti-diabetic and anti-cardiovascular disease effects, but its ability to protect against liver fibrosis has not been studied. Here, we assessed the ability of solubilized CoQ10 to improve dimethylnitrosamine (DMN)-induced liver fibrogenesis in mice. DMN treatments for 3 weeks produced a marked liver fibrosis as assessed by histopathological examination and tissue 4-hydroxyproline content. Solubilized CoQ10 (10 and 30 mg/kg) significantly inhibited both the increases in fibrosis score and 4-hydroxyproline content induced by DMN. Reverse transcription-polymerase chain reaction and Western blot analyses revealed that solubilized CoQ10 inhibitedmore » increases in the transforming growth factor-beta1 (TGF-beta1) mRNA and alpha-smooth muscle actin (alpha-SMA) protein by DMN. Interestingly, hepatic glutamate-cysteine ligase (GCL) and glutathione S-transferase A2 (GSTA2) were up-regulated in mice treated with CoQ10. Solubilized CoQ10 also up-regulated antioxidant enzymes such as catalytic subunits of GCL and GSTA2 via activating NF-E2 related factor2 (Nrf2)/antioxidant response element (ARE) in H4IIE hepatoma cells. Moreover, CoQ10's inhibition of alpha-SMA and TGF-beta1 expressions disappeared in Nrf2-null MEF cells. In contrast, Nrf2 overexpression significantly decreased the basal expression levels of alpha-SMA and TGF-beta1 in Nrf2-null MEF cells. These results demonstrated that solubilized CoQ10 inhibited DMN-induced liver fibrosis through suppression of TGF-beta1 expression via Nrf2/ARE activation.« less

The selective phosphorylation of a guanine nucleotide-binding regulatory protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carlson, K.E.

1989-01-01

Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the {alpha} subunit of G{sub i} and other G proteins in solution. However, the occurrence of the phosphorylation of G{sub 1} within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which themore » {alpha} subunits of G{sub i} undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with ({gamma}{sup 32}P)ATP and ({sup 32}P)H{sub 3}PO{sub 4}, respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G{sub i{alpha}}-despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G{sub z{alpha}}, or antibodies for both G{sub z{alpha}} and G{sub i{alpha}}, precipitated a 40-kDa phosphoprotein.« less

Decreased enzyme activity and contents of hepatic branched-chain alpha-keto acid dehydrogenase complex subunits in a rat model for type 2 diabetes mellitus.

PubMed

Bajotto, Gustavo; Murakami, Taro; Nagasaki, Masaru; Sato, Yuzo; Shimomura, Yoshiharu

2009-10-01

The mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC) is responsible for the committed step in branched-chain amino acid catabolism. In the present study, we examined BCKDC regulation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats both before (8 weeks of age) and after (25 weeks of age) the onset of type 2 diabetes mellitus. Long-Evans Tokushima Otsuka (LETO) rats were used as controls. Plasma branched-chain amino acid and branched-chain alpha-keto acid concentrations were significantly increased in young and middle-aged OLETF rats. Although the hepatic complex was nearly 100% active in all animals, total BCKDC activity and protein abundance of E1alpha, E1beta, and E2 subunits were markedly lower in OLETF than in LETO rats at 8 and 25 weeks of age. In addition, hepatic BCKDC activity and protein amounts were significantly decreased in LETO rats aged 25 weeks than in LETO rats aged 8 weeks. In skeletal muscle, E1beta and E2 proteins were significantly reduced, whereas E1alpha tended to increase in OLETF rats. Taken together, these results suggest that (1) whole-body branched-chain alpha-keto acid oxidation capacity is extremely reduced in OLETF rats independently of diabetes development, (2) the aging process decreases BCKDC activity and protein abundance in the liver of normal rats, and (3) differential posttranscriptional regulation for the subunits of BCKDC may exist in skeletal muscle.

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

PubMed Central

McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

1992-01-01

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes.

PubMed

Duperray, A; Berthier, R; Chagnon, E; Ryckewaert, J J; Ginsberg, M; Plow, E; Marguerie, G

1987-06-01

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.

Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L. IR-36).

PubMed

Seo, H S; Kim, H Y; Jeong, J Y; Lee, S Y; Cho, M J; Bahk, J D

1995-03-01

A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein alpha subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein alpha subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein alpha subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 microM [adenylate-32P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

1988-08-01

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost,more » a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.« less

The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc(1) Complex of Yeast Mitochondria.

PubMed

Conte, Laura; Zara, Vincenzo

2011-01-01

The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc(1) complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc(1) complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc(1) complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain.

The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc 1 Complex of Yeast Mitochondria

PubMed Central

Conte, Laura; Zara, Vincenzo

2011-01-01

The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc 1 complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc 1 complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc 1 complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain. PMID:21716720

Organocatalytic sequential alpha-amination-Horner-Wadsworth-Emmons olefination of aldehydes: enantioselective synthesis of gamma-amino-alpha,beta-unsaturated esters.

PubMed

Kotkar, Shriram P; Chavan, Vilas B; Sudalai, Arumugam

2007-03-15

A novel and highly enantioselective method for the synthesis of gamma-amino-alpha,beta-unsaturated esters via tandem alpha-amination-Horner-Wadsworth-Emmons (HWE) olefination of aldehydes is described. The one-pot assembly has been demonstrated for the construction of functionalized chiral 2-pyrrolidones, subunits present in several alkaloids. [structure: see text

Thyroid hormone is essential for pituitary somatotropes and lactotropes.

PubMed

Stahl, J H; Kendall, S K; Brinkmeier, M L; Greco, T L; Watkins-Chow, D E; Campos-Barros, A; Lloyd, R V; Camper, S A

1999-04-01

Mice homozygous for a disruption in the alpha-subunit essential for TSH, LH, and FSH activity (alphaGsu-/-) exhibit hypothyroidism and hypogonadism similar to that observed in TSH receptor-deficient hypothyroid mice (hyt) and GnRH-deficient hypogonadal mutants (hpg). Although the five major hormone-producing cells of the anterior pituitary are present in alphaGsu-/- mice, the relative proportions of each cell type are altered dramatically. Thyrotropes exhibit hypertrophy and hyperplasia, and somatotropes and lactotropes are underrepresented. The size and number of gonadotropes in alphaGsu mutants are not remarkable in contrast to the hypertrophy characteristic of gonadectomized animals. The reduction in lactotropes is more severe in alphaGsu mutants (13-fold relative to wild-type) than in hyt or hpg mutants (4.5- and 1.5-fold, respectively). In addition, T4 replacement therapy of alphaGsu mutants restores lactotropes to near-normal levels, illustrating the importance of T4, but not alpha-subunit, for lactotrope proliferation and function. T4 replacement is permissive for gonadotrope hypertrophy in alphaGsu mutants, consistent with the role for T4 in the function of gonadotropes. This study reveals the importance of thyroid hormone in developing the appropriate proportions of anterior pituitary cell types.

Steroid withdrawal in the mouse results in anxiogenic effects of 3alpha,5beta-THP: a possible model of premenstrual dysphoric disorder.

PubMed

Smith, Sheryl S; Ruderman, Yevgeniy; Frye, Cheryl; Homanics, Gregg; Yuan, Maoli

2006-06-01

3alpha-OH-5alpha[beta]-pregnan-20-one (THP) is a positive modulator of the GABAA receptor (GABAR), which underlies its reported anxiolytic effect. However, there are conditions such as premenstrual dysphoric disorder (PMDD) where increases in THP levels can be associated with adverse mood. In order to test for conditions where THP might be anxiogenic, we developed a mouse model of THP withdrawal. Because delta-containing GABAR are highly sensitive to THP modulation, results were compared in wild-type and delta knockout mice. Finasteride, a 5alpha-reductase blocker, was administered for 3 days to female wild-type or delta knockout mice. Then, animals were tested in the elevated plus maze, following acute administration of THP, lorazepam, flumazenil, or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), and results compared to vehicle-injected controls. CA1 hippocampal GABAR alpha4 subunit levels were assessed by Western blot. After THP withdrawal, THP produced anxiogenic effects, decreasing open arm entries on the elevated plus maze, following a brief shock, in contrast to its expected anxiolytic effects. As we have shown in rats, THP withdrawal also resulted in increased expression of the alpha4 subunit in mouse CA1 hippocampus. As expected for increases in alpha4-containing GABAR, THP withdrawn mice were relatively insensitive to the benzodiazepine (BDZ) lorazepam and had atypical responses to the BDZ antagonist flumazenil when tested on the plus maze. In contrast, they showed a greater anxiolytic response to THIP, which has greater efficacy at alpha4betadelta than other GABAR. Although THP withdrawal in delta knockout mice also increased the alpha4 GABAR subunit, the anxiogenic effects of THP and the anxiolytic effects of THIP were not observed, implicating alpha4betadelta GABAR in these effects. Based on these behavioral and pharmacological findings, we suggest that THP withdrawal in the mouse may serve as a rodent model of PMDD.

Improved purification of brine-shrimp (Artemia saline) (Na+ + K+)-activated adenosine triphosphatase and amino-acid and carbohydrate analyses of the isolated subunits.

PubMed Central

Peterson, G L; Hokin, L E

1980-01-01

Purification of the (Na+ + K+)-activated ATPase has been improved 2-fold the respect to both purity and yield over the previous method [Peterson, Ewing, Hootman & Conte (1978) J. Biol. Chem. 253, 4762-4770] by using Lubrol WX and non-denaturing concentrations of sodium dodecyl sulphate (SDS). The enzyme was purified 200-fold over the homogenate. The preparation had a specific activity of about 600 mumol of Pi/h per mg of protein, and was about 60% pure according to quantification of Coomassie Blue-stained SDS/polyacrylamide gels. The yield of purified enzyme was about 10 mg of protein per 100g of dry brine-shrimp (Artemia salina) cysts. The method is highly suitable for purification either on a small scale (10-25g of dry cysts) or on a large scale (900g of dry cysts) and methods are described for both. The large (Na+ + K+)-activated ATPase subunit (alpha-subunit) was isolated in pure form by SDS-gel filtration on Bio-Gel A 1.5m. The small subunit (beta-subunit) was eluted with other contaminating proteins on the Bio-Gel column, but was isolated in pure form by extraction from SDS/polyacrylamide gels. The amino acid and carbohydrate compositions of both subunits are reported. The alpha-subunit contained 5.2% carbohydrate by weight, and the beta-subunit 9.2%. Sialic acid was absent from both subunits. Images Fig. 3. Fig. 4. PMID:6272692

Role of the DELSEED Loop in Torque Transmission of F1-ATPase

PubMed Central

Tanigawara, Mizue; Tabata, Kazuhito V.; Ito, Yuko; Ito, Jotaro; Watanabe, Rikiya; Ueno, Hiroshi; Ikeguchi, Mitsunori; Noji, Hiroyuki

2012-01-01

F1-ATPase is an ATP-driven rotary motor that generates torque at the interface between the catalytic β-subunits and the rotor γ-subunit. The β-subunit inwardly rotates the C-terminal domain upon nucleotide binding/dissociation; hence, the region of the C-terminal domain that is in direct contact with γ—termed the DELSEED loop—is thought to play a critical role in torque transmission. We substituted all the DELSEED loop residues with alanine to diminish specific DELSEED loop-γ interactions and with glycine to disrupt the loop structure. All the mutants rotated unidirectionally with kinetic parameters comparable to those of the wild-type F1, suggesting that the specific interactions between DELSEED loop and γ is not involved in cooperative interplays between the catalytic β-subunits. Glycine substitution mutants generated half the torque of the wild-type F1, whereas the alanine mutant generated comparable torque. Fluctuation analyses of the glycine/alanine mutants revealed that the γ-subunit was less tightly held in the α3β3-stator ring of the glycine mutant than in the wild-type F1 and the alanine mutant. Molecular dynamics simulation showed that the DELSEED loop was disordered by the glycine substitution, whereas it formed an α-helix in the alanine mutant. Our results emphasize the importance of loop rigidity for efficient torque transmissions. PMID:23009846

Integrin expression and integrin-mediated adhesion in vitro of human multipotent stromal cells (MSCs) to endothelial cells from various blood vessels.

PubMed

Semon, Julie A; Nagy, Lauren H; Llamas, Claire B; Tucker, H Alan; Lee, Ryang Hwa; Prockop, Darwin J

2010-07-01

Multipotent mesenchymal stromal cells (MSCs) home to damaged tissue by processes partly regulated by integrins. Integrin subunits expressed by MSCs were identified by flow cytometry (FC), immunocytochemistry (IC), and a panel of integrin-binding antibodies. In subconfluent cultures, over 80% of MSCs expressed integrin subunits beta1, beta2, and alpha3, 20%-55% expressed alpha1, alpha2, alpha4, alpha5, alpha6, and alphaV, and about 10% expressed beta3 when assayed by FC. None of the cells expressed significant levels of 13 other integrins as assayed by FC, but seven of the 13 integrins were detected by IC: beta5, alpha7, alpha8, alpha9, alpha11, alphaX, and alphaD. Expression of some integrins changed with MSC confluency: integrins beta3, alpha1, alpha3, alpha5, and alphaV increased, and alpha6 decreased. Furthermore, alpha4 was the only integrin to vary among preparations of MSCs from different donors. The results resolved some discrepancies in the literature concerning integrin expression by MSCs. We also investigated the role of specific integrins in MSC adhesion to endothelial cells (ECs) from the pulmonary artery (HPAEC), cardiac-derived microvasculature (HMVEC-C), and umbilical veins (HUVEC). In experiments with blocking antibodies to beta integrins, anti-beta5 reduced MSC adhesion to all types of ECs, anti-beta1 to both HUVEC and HPAEC, anti-beta3 to HUVEC, and anti-beta2 to HMVEC-C. With blocking antibodies to alpha integrins, anti-alphaX reduced adhesion to HPAEC and HMVEC-C, anti-alphaV to HPAEC, and both anti-alpha7 and anti-alphaD to HMVEC-C. Thus, MSCs use diverse integrins to adhere to EC from various blood vessels in vitro.

Ca2+ signalling, voltage-gated Ca2+ channels and praziquantel in flatworm neuromusculature.

PubMed

Greenberg, R M

2005-01-01

Transient changes in calcium (Ca2+) levels regulate a wide variety of cellular processes, and cells employ both intracellular and extracellular sources of Ca2+ for signalling. Praziquantel, the drug of choice against schistosomiasis, disrupts Ca2+ homeostasis in adult worms. This review will focus on voltage-gated Ca2+ channels, which regulate levels of intracellular Ca2+ by coupling membrane depolarization to entry of extracellular Ca2+. Ca2+ channels are members of the ion channel superfamily and represent essential components of neurons, muscles and other excitable cells. Ca2+ channels are membrane protein complexes in which the pore-forming alpha1 subunit is modulated by auxiliary subunits such as beta and alpha2delta. Schistosomes express two Ca2+ channel beta subunit subtypes: a conventional subtype similar to beta subunits found in other vertebrates and invertebrates and a novel variant subtype with unusual structural and functional properties. The variant schistosome beta subunit confers praziquantel sensitivity to an otherwise praziquantel-insensitive mammalian Ca2+ channel, implicating it as a mediator of praziquantel action.

Clinical potential of meningioma genomic insights: a practical review for neurosurgeons.

PubMed

Karsy, Michael; Azab, Mohammed A; Abou-Al-Shaar, Hussam; Guan, Jian; Eli, Ilyas; Jensen, Randy L; Ormond, D Ryan

2018-06-01

Meningiomas are among the most common intracranial pathological conditions, accounting for 36% of intracranial lesions treated by neurosurgeons. Although the majority of these lesions are benign, the classical categorization of tumors by histological type or World Health Organization (WHO) grade has not fully captured the potential for meningioma progression and recurrence. Many targeted treatments have failed to generate a long-lasting effect on these tumors. Recently, several seminal studies evaluating the genomics of intracranial meningiomas have rapidly changed the understanding of the disease. The importance of NF2 (neurofibromin 2), TRAF7 (tumor necrosis factor [TNF] receptor-associated factor 7), KLF4 (Kruppel-like factor 4), AKT1, SMO (smoothened), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), and POLR2 (RNA polymerase II subunit A) demonstrates that there are at least 6 distinct mutational classes of meningiomas. In addition, 6 methylation classes of meningioma have been appreciated, enabling improved prediction of prognosis compared with traditional WHO grades. Genomic studies have shed light on the nature of recurrent meningioma, distinct intracranial locations and mutational patterns, and a potential embryonic cancer stem cell-like origin. However, despite these exciting findings, the clinical relevance of these findings remains elusive. The authors review the key findings from recent genomic studies in meningiomas, specifically focusing on how these findings relate to clinical insights for the practicing neurosurgeon.

Protein synthesis during acquisition of long-term facilitation is needed for the persistent loss of regulatory subunits of the Aplysia cAMP-dependent protein kinase.

PubMed Central

Bergold, P J; Sweatt, J D; Winicov, I; Weiss, K R; Kandel, E R; Schwartz, J H

1990-01-01

Depending on the number or the length of exposure, application of serotonin can produce either short-term or long-term presynaptic facilitation of Aplysia sensory-to-motor synapses. The cAMP-dependent protein kinase, a heterodimer of two regulatory and two catalytic subunits, has been shown to become stably activated only during long-term facilitation. Both acquisition of long-term facilitation and persistent activation of the kinase is blocked by anisomycin, an effective, reversible, and specific inhibitor of protein synthesis in Aplysia. We report here that 2-hr exposure of pleural sensory cells to serotonin lowers the concentration of regulatory subunits but does not change the concentration of catalytic subunits, as assayed 24 hr later; 5-min exposure to serotonin has no effect on either type of subunit. Increasing intracellular cAMP with a permeable analog of cAMP together with the phosphodiesterase inhibitor isobutyl methylxanthine also decreased regulatory subunits, suggesting that cAMP is the second messenger mediating serotonin action. Anisomycin blocked the loss of regulatory subunits only when applied with serotonin; application after the 2-hr treatment with serotonin had no effect. In the Aplysia accessory radula contractor muscle, prolonged exposure to serotonin or to the peptide transmitter small cardioactive peptide B, both of which produce large increases in intracellular cAMP, does not decrease regulatory subunits. This mechanism of regulating the cAMP-dependent protein kinase therefore may be specific to the nervous system. We conclude that during long-term facilitation, new protein is synthesized in response to the facilitatory stimulus, which changes the ratio of subunits of the cAMP-dependent protein kinase. This alteration in ratio could persistently activate the kinase and produce the persistent phosphorylation seen in long-term facilitated sensory cells. Images PMID:1692622

Protein kinase A activation: Something new under the sun?

PubMed

Smith, F Donelson; Scott, John D

2018-05-17

The role of autophosphorylation of the type II regulatory subunit in activation of protein kinase A (PKA) has been a longstanding question. In this issue, Isensee et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201708053) use antibody tools that selectively recognize phosphorylated RII and the catalytic subunit active site to reexamine PKA holoenzyme activation mechanisms in neurons. © 2018 Smith and Scott.

Probing the importance of the hemilabile site of bis(phosphine) monoxide ligands in the copper-catalyzed addition of diethylzinc to N-phosphinoylimines: discovery of new effective chiral ligands.

PubMed

Bonnaventure, Isabelle; Charette, André B

2008-08-15

The hemilabile ligand Me-DuPHOS(O) 2 has proven to be a successful ligand for the copper-catalyzed addition of diethylzinc to N-phosphinoylimines. The corresponding alpha-chiral amines were obtained in high yields (80-98%) and enantiomeric ratios (19.0:1 to 99.0:1 er). Furthermore, this Cu* 2 catalytic system has been shown to be effective in the addition of diethylzinc to nitroalkenes and in the reduction of beta,beta-disubstituted vinyl phenyl sulfones. This paper describes a general structure/selectivity study in which the three ligand subunits (chiral phospholane-linker-labile coordinating group (Z)) are systematically modified and tested in the copper-catalyzed addition of diethylzinc to the N-phosphinoylimine 1 derived from benzaldehyde. This study led to the discovery of a new class of effective chiral ligands that combine a chiral phospholane unit and an achiral phosphine oxide.

The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species.

PubMed

Liu, Sidong; Charlesworth, Thomas J; Bason, John V; Montgomery, Martin G; Harbour, Michael E; Fearnley, Ian M; Walker, John E

2015-05-15

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.

The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species

PubMed Central

Liu, Sidong; Charlesworth, Thomas J.; Bason, John V.; Montgomery, Martin G.; Harbour, Michael E.; Fearnley, Ian M.; Walker, John E.

2015-01-01

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex. PMID:25759169

IκB kinaseα/β control biliary homeostasis and hepatocarcinogenesis in mice by phosphorylating the cell-death mediator receptor-interacting protein kinase 1.

PubMed

Koppe, Christiane; Verheugd, Patricia; Gautheron, Jérémie; Reisinger, Florian; Kreggenwinkel, Karina; Roderburg, Christoph; Quagliata, Luca; Terracciano, Luigi; Gassler, Nikolaus; Tolba, René H; Boege, Yannick; Weber, Achim; Karin, Michael; Luedde, Mark; Neumann, Ulf P; Weiskirchen, Ralf; Tacke, Frank; Vucur, Mihael; Trautwein, Christian; Lüscher, Bernhard; Preisinger, Christian; Heikenwalder, Mathias; Luedde, Tom

2016-10-01

The IκB-Kinase (IKK) complex-consisting of the catalytic subunits, IKKα and IKKβ, as well as the regulatory subunit, NEMO-mediates activation of the nuclear factor κB (NF-κB) pathway, but previous studies suggested the existence of NF-κB-independent functions of IKK subunits with potential impact on liver physiology and disease. Programmed cell death is a crucial factor in the progression of liver diseases, and receptor-interacting kinases (RIPKs) exerts strategic control over multiple pathways involved in regulating novel programmed cell-death pathways and inflammation. We hypothesized that RIPKs might be unrecognized targets of the catalytic IKK-complex subunits, thereby regulating hepatocarcinogenesis and cholestasis. In this present study, mice with specific genetic inhibition of catalytic IKK activity in liver parenchymal cells (LPCs; IKKα/β(LPC-KO) ) were intercrossed with RIPK1(LPC-KO) or RIPK3(-/-) mice to examine whether RIPK1 or RIPK3 might be downstream targets of IKKs. Moreover, we performed in vivo phospho-proteome analyses and in vitro kinase assays, mass spectrometry, and mutagenesis experiments. These analyses revealed that IKKα and IKKβ-in addition to their known function in NF-κB activation-directly phosphorylate RIPK1 at distinct regions of the protein, thereby regulating cell viability. Loss of this IKKα/β-dependent RIPK1 phosphorylation in LPCs inhibits compensatory proliferation of hepatocytes and intrahepatic biliary cells, thus impeding HCC development, but promoting biliary cell paucity and lethal cholestasis. IKK-complex subunits transmit a previously unrecognized signal through RIPK1, which is fundamental for the long-term consequences of chronic hepatic inflammation and might have potential implications for future pharmacological strategies against cholestatic liver disease and cancer. (Hepatology 2016;64:1217-1231). © 2016 by the American Association for the Study of Liver Diseases.

Developmentally-regulated sodium channel subunits are differentially sensitive to {alpha}-cyano containing pyrethroids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meacham, Connie A.; Brodfuehrer, Peter D.; Watkins, Jennifer A.

2008-09-15

Juvenile rats have been reported to be more sensitive to the acute neurotoxic effects of the pyrethroid deltamethrin than adults. While toxicokinetic differences between juveniles and adults are documented, toxicodynamic differences have not been examined. Voltage-gated sodium channels, the primary targets of pyrethroids, are comprised of {alpha} and {beta} subunits, each of which have multiple isoforms that are expressed in a developmentally-regulated manner. To begin to test whether toxicodynamic differences could contribute to age-dependent deltamethrin toxicity, deltamethrin effects were examined on sodium currents in Xenopus laevis oocytes injected with different combinations of rat {alpha} (Na{sub v}1.2 or Na{sub v}1.3) andmore » {beta} ({beta}{sub 1} or {beta}{sub 3}) subunits. Deltamethrin induced tail currents in all isoform combinations and increased the percent of modified channels in a concentration-dependent manner. Effects of deltamethrin were dependent on subunit combination; Na{sub v}1.3-containing channels were modified to a greater extent than were Na{sub v}1.2-containing channels. In the presence of a {beta} subunit, deltamethrin effects were significantly greater, an effect most pronounced for Na{sub v}1.3 channels; Na{sub v}1.3/{beta}{sub 3} channels were more sensitive to deltamethrin than Na{sub v}1.2/{beta}{sub 1} channels. Na{sub v}1.3/{beta}{sub 3} channels are expressed embryonically, while the Na{sub v}1.2 and {beta}{sub 1} subunits predominate in adults, supporting the hypothesis for age-dependent toxicodynamic differences. Structure-activity relationships for sensitivity of these subunit combinations were examined for other pyrethroids. Permethrin and tetramethrin did not modify currents mediated by either subunit combination. Cypermethrin, {beta}-cyfluthrin, esfenvalerate and fenpropathrin all modified sodium channel function; effects were significantly greater on Na{sub v}1.3/{beta}{sub 3} than on Na{sub v}1.2/{beta}{sub 1} channels. These data demonstrate a greater sensitivity of Na{sub v}1.3 vs Na{sub v}1.2 channels to deltamethrin and other cyano-containing pyrethroids, particularly in the presence of a {beta} subunit.« less

Structure of the aspartic protease from Rous sarcoma retrovirus refined at 2-A resolution.

PubMed

Jaskólski, M; Miller, M; Rao, J K; Leis, J; Wlodawer, A

1990-06-26

The structure of Rous sarcoma virus protease has been solved by multiple isomorphous replacement in the crystal form belonging to space group P3(1)21, with unit-cell parameters a = 88.95 A and c = 78.90 A. The enzyme belongs to the family of aspartic proteases with two identical subunits composing the active homodimer. The noncrystallographic dyad relating these two subunits was identified after preliminary tracing in the MIR map and was used for phase improvement by electron-density averaging. Structure refinement resulted in a model that included 1772 protein atoms and 252 water molecules, with an R factor of 0.144 for data extending to 2 A. The secondary structure of a retroviral protease molecule closely resembles that of a single domain in pepsin-like aspartic proteases and consists of several beta-strands and of one well-defined and one distorted alpha-helix. The dimer interface is composed of the N- and C-terminal chains from both subunits which are intertwined to form a well-ordered four-stranded antiparallel beta-sheet. In each monomer, the catalytic triad (Asp-Ser-Gly) is located in a loop that forms a part of the psi-structure characteristic to all aspartic proteases. The position of a water molecule between the active-site aspartate residues and the general scheme of H bonding within the active site bear close resemblance to those in pepsin-like aspartic proteases and therefore suggest a similar enzymatic mechanism. The binding cleft over the active site is covered by two flap arms, one from each monomer, which are partially disordered. The retroviral protease dimer has been compared with several enzymes of cellular origin, with chains aligning to an rms deviation of 1.90 A or better.

Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme.

PubMed

Cho, Uhn Soo; Xu, Wenqing

2007-01-04

Protein phosphatase 2A (PP2A) is a principal Ser/Thr phosphatase, the deregulation of which is associated with multiple human cancers, Alzheimer's disease and increased susceptibility to pathogen infections. How PP2A is structurally organized and functionally regulated remains unclear. Here we report the crystal structure of an AB'C heterotrimeric PP2A holoenzyme. The structure reveals that the HEAT repeats of the scaffold A subunit form a horseshoe-shaped fold, holding the catalytic C and regulatory B' subunits together on the same side. The regulatory B' subunit forms pseudo-HEAT repeats and interacts with the C subunit near the active site, thereby defining substrate specificity. The methylated carboxy-terminal tail of the C subunit interacts with a highly negatively charged region at the interface between A and B' subunits, suggesting that the C-terminal carboxyl methylation of the C subunit promotes B' subunit recruitment by neutralizing charge repulsion. Together, our structural results establish a crucial foundation for understanding PP2A assembly, substrate recruitment and regulation.

Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene

NASA Technical Reports Server (NTRS)

Yu, Y.; Okayasu, R.; Weil, M. M.; Silver, A.; McCarthy, M.; Zabriskie, R.; Long, S.; Cox, R.; Ullrich, R. L.

2001-01-01

Female BALB/c mice are unusually radiosensitive and more susceptible than C57BL/6 and other tested inbred mice to ionizing radiation (IR)-induced mammary tumors. This breast cancer susceptibility is correlated with elevated susceptibility for mammary cell transformation and genomic instability following irradiation. In this study, we report the identification of two BALB/c strain-specific polymorphisms in the coding region of Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit, which is known to be involved in DNA double-stranded break repair and post-IR signal transduction. First, we identified an A --> G transition at base 11530 resulting in a Met --> Val conversion at codon 3844 (M3844V) in the phosphatidylinositol 3-kinase domain upstream of the scid mutation (Y4046X). Second, we identified a C --> T transition at base 6418 resulting in an Arg --> Cys conversion at codon 2140 (R2140C) downstream of the putative leucine zipper domain. This unique PrkdcBALB variant gene is shown to be associated with decreased DNA-dependent protein kinase catalytic subunit activity and with increased susceptibility to IR-induced genomic instability in primary mammary epithelial cells. The data provide the first evidence that naturally arising allelic variation in a mouse DNA damage response gene may associate with IR response and breast cancer risk.

Icaritin requires Phosphatidylinositol 3 kinase (PI3K)/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading

PubMed Central

ZHANG, Zong-Kang; LI, Jie; LIU, Jin; GUO, Baosheng; LEUNG, Albert; ZHANG, Ge; ZHANG, Bao-Ting

2016-01-01

Counteracting muscle atrophy induced by mechanical unloading/inactivity is of great clinical need and challenge. A therapeutic agent that could counteract muscle atrophy following mechanical unloading in safety is desired. This study showed that natural product Icaritin (ICT) could increase the phosphorylation level of Phosphatidylinositol 3 kinase (PI3K) at p110 catalytic subunit and promote PI3K/Akt signaling markers in C2C12 cells. This study further showed that the high dose ICT treatment could significantly attenuate the decreases in the phosphorylation level of PI3K at p110 catalytic subunit and its downstream markers related to protein synthesis, and inhibit the increases in protein degradation markers at mRNA and protein levels in rat soleus muscle following 28-day hindlimb unloading. In addition, the decreases in soleus muscle mass, muscle fiber cross-sectional area, twitch force, specific force, contraction time and half relaxation time could be significantly attenuated by the high dose ICT treatment. The low dose ICT treatment could moderately attenuate the above changes induced by unloading. Wortmannin, a specific inhibitor of PI3K at p110 catalytic subunit, could abolish the above effects of ICT in vitro and in vivo, indicating that PI3K/Akt signaling could be required by ICT to counteract skeletal muscle atrophy following mechanical unloading. PMID:26831566

A synthetic peptide derived from A1 module in CRD4 of human TNF receptor-1 inhibits binding and proinflammatory effect of human TNF-alpha.

PubMed

Cao, Yingnan; Wang, Zhaohe; Bu, Xianzhang; Tang, Shu; Mei, Zhengrong; Liu, Peiqing

2009-06-01

Tumour necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine, which has been shown to be a causative factor in rheumatoid arthritis, inflammatory bowel disease and septic shock. Proinflammatory effect of TNF-alpha is activated mainly through human TNF receptor-1 (TNF-R1). However, the role of the fourth cystein-rich domain (CRD4) of TNF-R1 extracellular portion in the interaction of TNF-alpha with TNF-R1 is still unclear. In the present study, binding activity of TNF-alpha to TNF-R1 and protein levels of IkappaB-alpha and nuclear transcription factor kappa B (NF-kappaB) p65 subunit in HeLa cells were measured using enzyme-linked immunosorbent assay (ELISA) and western-blot analysis. Pep 3 (LRENECVS) which was derived from the hydrophilic region of A1 module in CRD4 remarkably inhibited the binding of TNF-alpha to TNF-R1, and also reversed TNF-alpha-induced degradation of IkappaB-alpha and nuclear translocation of NF-kappaB p65 subunit in HeLa cells. Our results confirmed that the hydrophilic region of A1 module in CRD4 participated in the interaction of TNF-alpha with TNF-R1, and demonstrated the potential of small-molecule TNF-alpha extracellular inhibitors targeting at A1 module in CRD4 of TNF-R1 in suppressing proinflammatory effect of TNF-alpha.

Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells.

PubMed

Prulière-Escabasse, Virginie; Planès, Carole; Escudier, Estelle; Fanen, Pascale; Coste, André; Clerici, Christine

2007-11-23

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.

Maxi- and mini-ferritins: minerals and protein nanocages.

PubMed

Bevers, Loes E; Theil, Elizabeth C

2011-01-01

Ferritins synthesize ferric oxide biominerals and are central to all life for concentrating iron and protection against oxidative stress from the ferrous and oxidant chemistry. The ferritin protein nanocages and biomineral synthesis are discussed in terms of wide biological distribution of the maxi-ferritins (24 subunit ± heme) and mini-ferritins (Dps) (12 subunit), conservations of the iron/oxygen catalytic sites in the protein cages, mineral formation (step i. Fe(II) entry and binding, step ii. O(2) or H(2)O(2) binding and formation of transition intermediates, step iii. release of differric oxo mineral precursors from active sites, step iv. nucleation and mineralization) properties of the minerals, and protein control of mineral dissolution and release of Fe(II). Pores in ferritin protein cages control iron entry for mineralization and iron exit after mineral dissolution. The relationship between phosphate or the presence of catalytically inactive subunits (animal L subunits) and ferritin iron mineral disorder is developed based on new information about contributions of ferritin protein cage structure to nucleation in protein cage subunit channels that exit close enough to those of other subunits and exiting mineral nuclei to facilitate bulk mineral formation. How and where protons move in and out of the protein during mineral synthesis and dissolution, how ferritin cage assembly with 12 or 24 subunits is encoded in the widely divergent ferritin amino acid sequences, and what is the role of the protein in synthesis of the bulk mineral are all described as problems requiring new approaches in future investigations of ferritin biominerals.

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis.

PubMed

Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Mélida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen

2013-10-29

Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200-300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP.

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis

PubMed Central

Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Mélida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen

2013-01-01

Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200–300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP. PMID:24127606

Effect of chronic hypokalemia on H(+)-K(+)-ATPase expression in rat colon.

PubMed

Codina, J; Pressley, T A; DuBose, T D

1997-01-01

Although the kidney plays the major role in the regulation of systemic K+ homeostasis, the colon also participates substantively in K+ balance. The colon is capable of both K+ absorption and secretion, the magnitude of which can be modulated in response to dietary K+ intake. The H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) has been proposed as a possible mediator of K+ absorption in distal colon, but inhibitor profiles obtained in recent studies suggest that two, and perhaps more, distinct H(+)-K(+)-ATPase activities may be present in mammalian distal colon. We have developed highly specific probes for the catalytic alpha-subunits of colonic and gastric H(+)-K(+)-ATPase, alpha 1-Na(+)-K(+)-ATPase, and beta-actin, which were used in Northern analysis of total RNA from whole distal colon and stomach obtained from one of three experimental groups of rats: 1) controls, 2) chronic dietary K+ depletion, and 3) chronic metabolic acidosis. The probe for the colonic but not the gastric H(+)-K(+)-ATPase alpha-isoform hybridized to distal colon total RNA in all groups. A significant increase in colonic H(+)-K(+)-ATPase mRNA abundance was observed in response to chronic dietary K+ depletion but not to chronic metabolic acidosis. The alpha 1-isoform of Na(+)-K(+)-ATPase, which is also expressed in distal colon, did not respond consistently to either chronic dietary K+ depletion or chronic metabolic acidosis. The gastric probe did not hybridize to total RNA from distal colon but, as expected, hybridized to total stomach RNA. However, the abundance of gastric H(+)-K(+)-ATPase or Na(+)-K(+)-ATPase in stomach was not altered consistently by either chronic dietary K+ depletion or metabolic acidosis. Under the conditions of this study, it appears that the mRNA encoding the colonic alpha-isoform is upregulated by chronic dietary K+ restriction, a condition shown previously to increase K+ absorption in the distal colon.

Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

PubMed

Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M

1996-11-01

It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

Regulation of acetylcholine receptor alpha subunit variants in human myasthenia gravis. Quantification of steady-state levels of messenger RNA in muscle biopsy using the polymerase chain reaction.

PubMed Central

Guyon, T; Levasseur, P; Truffault, F; Cottin, C; Gaud, C; Berrih-Aknin, S

1994-01-01

Myasthenia gravis (MG) is an autoimmune disease mediated by auto-antibodies that attack the nicotinic acetylcholine receptor (AChR). To elucidate the molecular mechanisms underlying the decrease in AChR levels at the neuromuscular junction, we investigated the regulation of AChR expression by analyzing mRNA of the two AChR alpha subunit isoforms (P3A+ and P3A-) in muscle samples from myasthenic patients relative to controls. We applied a quantitative method based on reverse transcription of total RNA followed by polymerase chain reaction (PCR), using an internal standard we constructed by site-directed mutagenesis. An increased expression of mRNA coding for the alpha subunit of the AChR isoforms was observed in severely affected patients (P < 0.003 versus controls) but not in moderately affected patients, independently of the anti-AChR antibody titer. Study of mRNA precursor levels indicates a higher expression in severely affected patients compared to controls, suggesting an enhanced rate of transcription of the message coding for the alpha subunit isoforms in these patients. We have also reported that mRNA encoding both isoforms are expressed at an approximate 1:1 ratio in controls and in patients. We have thus identified a new biological parameter correlated with disease severity, and provide evidence of a compensatory mechanism to balance the loss of AChR in human myasthenia gravis, which is probably triggered only above a certain degree of AChR loss. Images PMID:8040257

Molecular study of electron transfer flavoprotein alpha-subunit deficiency in two Japanese children with different phenotypes of glutaric acidemia type II.

PubMed

Purevjav, E; Kimura, M; Takusa, Y; Ohura, T; Tsuchiya, M; Hara, N; Fukao, T; Yamaguchi, S

2002-09-01

Electron transfer flavoprotein is a mitochondrial matrix protein composed of alpha- and beta-subunits (ETF alpha and ETF beta, respectively). This protein transfers electrons between several mitochondrial dehydrogenases and the main respiratory chain via ETF dehydrogenase (ETF-DH). Defects in ETF or ETF-DH cause glutaric acidemias type II (GAII). We investigated the molecular basis of ETF alpha deficiency in two Japanese children with different clinical phenotypes using expression study. Patient 1 had the severe form of GAII, a compound heterozygote of two mutations: 799G to A (alpha G267R) and nonsense 7C to T (alpha R3X). Patient 2 had the mild form and carried two heterozygous mutations: 764G to T (alpha G255V) and 478delG (frameshift). Both patients had one each of missense mutations in one allele; the others were either nonsense or truncated. Restriction enzyme digestion assay using genomic DNAs from 100 healthy Japanese revealed that these mutations were all novel. No signal for ETF alpha was detected by immunoblotting in cases of missense mutants, while wild-type cDNA resulted in expression of ETF alpha protein. Transfection with wild-type ETF alpha cDNA into cultured cells from both patients elevated incorporation of radioisotope-labelled fatty acids. These four mutations were pathogenic for GAII and missense mutations, alpha G255V and alpha G267R were considered anecdotal for mild and severe forms, respectively.

Regulation of the PI3K pathway through a p85α monomer–homodimer equilibrium | Office of Cancer Genomics

Cancer.gov

The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity.

HupW Protease Specifically Required for Processing of the Catalytic Subunit of the Uptake Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120

PubMed Central

Lindberg, Pia; Devine, Ellenor; Stensjö, Karin

2012-01-01

The maturation process of [NiFe] hydrogenases includes a proteolytic cleavage of the large subunit. We constructed a mutant of Nostoc strain PCC 7120 in which hupW, encoding a putative hydrogenase-specific protease, is inactivated. Our results indicate that the protein product of hupW selectively cleaves the uptake hydrogenase in this cyanobacterium. PMID:22020512

Roles of human POLD1 and POLD3 in genome stability

PubMed Central

Tumini, Emanuela; Barroso, Sonia; -Calero, Carmen Pérez; Aguilera, Andrés

2016-01-01

DNA replication is essential for cellular proliferation. If improperly controlled it can constitute a major source of genome instability, frequently associated with cancer and aging. POLD1 is the catalytic subunit and POLD3 is an accessory subunit of the replicative Pol δ polymerase, which also functions in DNA repair, as well as the translesion synthesis polymerase Pol ζ, whose catalytic subunit is REV3L. In cells depleted of POLD1 or POLD3 we found a differential but general increase in genome instability as manifested by DNA breaks, S-phase progression impairment and chromosome abnormalities. Importantly, we showed that both proteins are needed to maintain the proper amount of active replication origins and that POLD3-depletion causes anaphase bridges accumulation. In addition, POLD3-associated DNA damage showed to be dependent on RNA-DNA hybrids pointing toward an additional and specific role of this subunit in genome stability. Interestingly, a similar increase in RNA-DNA hybrids-dependent genome instability was observed in REV3L-depleted cells. Our findings demonstrate a key role of POLD1 and POLD3 in genome stability and S-phase progression revealing RNA-DNA hybrids-dependent effects for POLD3 that might be partly due to its Pol ζ interaction. PMID:27974823

Regulated assembly and disassembly of the yeast telomerase quaternary complex

PubMed Central

Tucey, Timothy M.

2014-01-01

The enzyme telomerase, which elongates chromosome termini, is a critical factor in determining long-term cellular proliferation and tissue renewal. Hence, even small differences in telomerase levels can have substantial consequences for human health. In budding yeast, telomerase consists of the catalytic Est2 protein and two regulatory subunits (Est1 and Est3) in association with the TLC1 RNA, with each of the four subunits essential for in vivo telomerase function. We show here that a hierarchy of assembly and disassembly results in limiting amounts of the quaternary complex late in the cell cycle, following completion of DNA replication. The assembly pathway, which is driven by interaction of the Est3 telomerase subunit with a previously formed Est1–TLC1–Est2 preassembly complex, is highly regulated, involving Est3-binding sites on both Est2 and Est1 as well as an interface on Est3 itself that functions as a toggle switch. Telomerase subsequently disassembles by a mechanistically distinct pathway due to dissociation of the catalytic subunit from the complex in every cell cycle. The balance between the assembly and disassembly pathways, which dictate the levels of the active holoenzyme in the cell, reveals a novel mechanism by which telomerase (and hence telomere homeostasis) is regulated. PMID:25240060

Subunit III-depleted cytochrome c oxidase provides insight into the process of proton uptake by proteins

PubMed Central

Varanasi, Lakshman; Hosler, Jonathan P.

2011-01-01

We review studies of subunit III-depleted cytochrome c oxidase (CcO III (−)) that elucidate the structural basis of steady-state proton uptake from solvent into an internal proton transfer pathway. The removal of subunit III from R. sphaeroides CcO makes proton uptake into the D pathway a rate-determining step, such that measurements of the pH dependence of steady-state O2 consumption can be used to compare the rate and functional pKa of proton uptake by D pathways containing different initial proton acceptors. The removal of subunit III also promotes spontaneous suicide inactivation by CcO, greatly shortening its catalytic lifespan. Because the probability of suicide inactivation is controlled by the rate at which the D pathway delivers protons to the active site, measurements of catalytic lifespan provide a second method to compare the relative efficacy of proton uptake by engineered CcO III (−) forms. These simple experimental systems have been used to explore general questions of proton uptake by proteins, such as the functional value of an initial proton acceptor, whether an initial acceptor must be surface-exposed, which side chains will function as initial proton acceptors and whether multiple acceptors can speed proton uptake. PMID:22023935

Inhibitory function of adapter-related protein complex 2 alpha 1 subunit in the process of nuclear translocation of human immunodeficiency virus type 1 genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kitagawa, Yukiko; Department of Oral and Maxillofacial Surgery II, Osaka University, Osaka 565-0871; Kameoka, Masanori

2008-03-30

The transfection of human cells with siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2{alpha}) was revealed to significantly up-regulate the replication of human immunodeficiency virus type 1 (HIV-1). This effect was confirmed by cell infection with vesicular stomatitis virus G protein-pseudotyped HIV-1 as well as CXCR4-tropic and CCR5-tropic HIV-1. Viral adsorption, viral entry and reverse transcription processes were not affected by cell transfection with siRNA against AP2{alpha}. In contrast, viral nuclear translocation as well as the integration process was significantly up-regulated in cells transfected with siRNA against AP2{alpha}. Confocal fluorescence microscopy revealed that a subpopulation of AP2{alpha} wasmore » not only localized in the cytoplasm but was also partly co-localized with lamin B, importin {beta} and Nup153, implying that AP2{alpha} negatively regulates HIV-1 replication in the process of nuclear translocation of viral DNA in the cytoplasm or the perinuclear region. We propose that AP2{alpha} may be a novel target for disrupting HIV-1 replication in the early stage of the viral life cycle.« less

Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

PubMed Central

Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

1992-01-01

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

Lactobacillus cypricasei Lawson et al. 2001 is a later heterotypic synonym of Lactobacillus acidipiscis Tanasupawat et al. 2000.

PubMed

Naser, Sabri M; Vancanneyt, Marc; Hoste, Bart; Snauwaert, Cindy; Swings, Jean

2006-07-01

The applicability of a multilocus sequence analysis (MLSA)-based identification system for lactobacilli was evaluated. Two housekeeping genes that code for the phenylalanyl-tRNA synthase alpha-subunit (pheS) and RNA polymerase alpha-subunit (rpoA) were sequenced and analysed for members of the Lactobacillus salivarius species group. The type strains of Lactobacillus acidipiscis and Lactobacillus cypricasei were investigated further using a third gene that encodes the alpha-subunit of ATP synthase (atpA). The MLSA data revealed close relatedness between L. acidipiscis and L. cypricasei, with 99.8-100 % pheS, rpoA and atpA gene sequence similarities. Comparison of the 16S rRNA gene sequences of the type strains of the two species confirmed the close relatedness (99.8 % gene sequence similarity) between the two taxa. Similar phenotypes and high DNA-DNA binding values in the range of 84 to 97.5 % confirmed that L. acidipiscis and L. cypricasei are synonymous species. On the basis of the present study, it is proposed that Lactobacillus cypricasei is a later heterotypic synonym of Lactobacillus acidipiscis.

Selective labelling of diazepam-insensitive GABAA receptors in vivo using [3H]Ro 15-4513.

PubMed

Pym, Luanda J; Cook, Susan M; Rosahl, Thomas; McKernan, Ruth M; Atack, John R

2005-11-01

Classical benzodiazepines (BZs), such as diazepam, bind to GABAA receptors containing alpha1, alpha2, alpha3 or alpha5 subunits that are therefore described as diazepam-sensitive (DS) receptors. However, the corresponding binding site of GABAA receptors containing either an alpha4 or alpha6 subunit do not bind the classical BZs and are therefore diazepam-insensitive (DIS) receptors; a difference attributable to a single amino acid (histidine in alpha1, alpha2, alpha3 and alpha5 subunits and arginine in alpha4 and alpha6). Unlike classical BZs, the imidazobenzodiazepines Ro 15-4513 and bretazenil bind to both DS and DIS populations of GABAA receptors. In the present study, an in vivo assay was developed using lorazepam to fully occupy DS receptors such that [3H]Ro 15-4513 was then only able to bind to DIS receptors. When dosed i.v., [3H]Ro 15-4513 rapidly entered and was cleared from the brain, with approximately 70% of brain radioactivity being membrane-bound. Essentially all membrane binding to DS+DIS receptors could be displaced by unlabelled Ro 15-4513 or bretazenil, with respective ID50 values of 0.35 and 1.2 mg kg(-1). A dose of 30 mg kg(-1) lorazepam was used to block all DS receptors in a [3H]Ro 15-1788 in vivo binding assay. When predosed in a [3H]Ro 15-4513 binding assay, lorazepam blocked [3H]Ro 15-4513 binding to DS receptors, with the remaining binding to DIS receptors accounting for 5 and 23% of the total (DS plus DIS) receptors in the forebrain and cerebellum, respectively. The in vivo binding of [3H]Ro 15-4513 to DIS receptors in the presence of lorazepam was confirmed using alpha1H101R knock-in mice, in which alpha1-containing GABAA receptors are rendered diazepam insensitive by mutation of the histidine that confers diazepam sensitivity to arginine. In these mice, and in the presence of lorazepam, there was an increase of in vivo [3H]Ro 15-4513 binding in the forebrain and cerebellum from 4 and 15% to 36 and 59% of the total (i.e. DS plus DIS) [3H]Ro 15-4513 binding observed in the absence of lorazepam.

alpha 4 beta 2 subunit combination specific pharmacology of neuronal nicotinic acetylcholine receptors in N1E-115 neuroblastoma cells.

PubMed

Zwart, R; Abraham, D; Oortgiesen, M; Vijverberg, H P

1994-08-22

Pharmacological characteristics of native neuronal nicotinic acetylcholine receptor-mediated ion currents in mouse N1E-115 neuroblastoma cells have been investigated by superfusion of voltage clamped cells with known concentrations of the agonists acetylcholine, nicotine and cytisine, and the antagonists alpha-bungarotoxin and neuronal bungarotoxin. The sensitivity of the nicotinic acetylcholine receptor for agonists followed the agonist potency rank-order: nicotine approximately acetylcholine > cytisine. The EC50 values of acetylcholine and nicotine are 78 microM and 76 microM, respectively. Equal concentrations of acetylcholine and nicotine induce inward currents with approximately the same peak amplitude whereas cytisine induces much smaller inward currents. Acetylcholine-induced currents are unaffected by high concentrations of alpha-bungarotoxin. Conversely, at 10 and 90 nM neuronal bungarotoxin reduces the amplitude of the 1 mM acetylcholine-induced inward current to 47% and 11% of control values, respectively. Both the agonist potency rank-order and the differential sensitivity to snake toxins of nicotinic receptors in N1E-115 cells are consistent with the known pharmacological profile of alpha 4 beta 2 nicotinic receptors expressed in Xenopus oocytes and distinct from those of all other nicotinic acetylcholine receptors of known functional subunit compositions. All data indicate that the native nicotinic acetylcholine receptor in N1E-115 cells is an assembly of alpha 4 and beta 2 subunits, the putative major subtype of nicotinic acetylcholine receptor in the brain.

Design Strategy of Multi-electron Transfer Catalysts Based on a Bioinformatic Analysis of Oxygen Evolution and Reduction Enzymes.

PubMed

Ooka, Hideshi; Hashimoto, Kazuhito; Nakamura, Ryuhei

2018-05-14

Understanding the design strategy of photosynthetic and respiratory enzymes is important to develop efficient artificial catalysts for oxygen evolution and reduction reactions. Here, based on a bioinformatic analysis of cyanobacterial oxygen evolution and reduction enzymes (photosystem II: PS II and cytochrome c oxidase: COX, respectively), the gene encoding the catalytic D1 subunit of PS II was found to be expressed individually across 38 phylogenetically diverse strains, which is in contrast to the operon structure of the genes encoding major COX subunits. Selective synthesis of the D1 subunit minimizes the repair cost of PS II, which allows compensation for its instability by lowering the turnover number required to generate a net positive energy yield. The different bioenergetics observed between PS II and COX suggest that in addition to the catalytic activity rationalized by the Sabatier principle, stability factors have also provided a major influence on the design strategy of biological multi-electron transfer enzymes. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Neurospora crassa alpha-ketoglutarate dehydrogenase complex: description, resolution of components and catalytic properties.

PubMed

Bessam, H; Mareck, A M; Foucher, B

1989-01-27

A method is proposed for the purification of the Neurospora crassa alpha-ketoglutarate dehydrogenase complex, and the main points for preserving its activity, which seems to be particularly fragile in fungus, are discussed. Resolution of the constitutive enzymes was attempted and permitted the identification of the three protein bands resolved on SDS-polyacrylamide gel electrophoresis as E3, E1 and E2 with respective Mr values of 54,000, 53,000 and 49,000. Catalytic properties of the purified complex were established showing the importance of divalent cations in regulating the activity level. The role of Ca2+ in particular was investigated. It was shown that Ca2+ diminishes the Km value of the N. crassa alpha-ketoglutarate dehydrogenase complex for alpha-ketoglutarate in the physiological concentration range, as previously observed for the mammalian complexes.

Preparation and catalytic activities of LaFeO3 and Fe2O3 for HMX thermal decomposition.

PubMed

Wei, Zhi-Xian; Xu, Yan-Qing; Liu, Hai-Yan; Hu, Chang-Wen

2009-06-15

Perovskite-type LaFeO(3) and alpha-Fe(2)O(3) with high specific surface areas were directly prepared with appropriate stearic acid-nitrates ratios by a novel stearic acid solution combustion method. The obtained powders were characterized by XRD, FT-IR and XPS techniques. The catalytic activities of perovskite-type LaFeO(3) and alpha-Fe(2)O(3) for the thermal decomposition of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) were investigated by TG and TG-EGA techniques. The experimental results show that the catalytic activity of perovskite-type LaFeO(3) was much higher than that of alpha-Fe(2)O(3) because of higher concentration of surface-adsorbed oxygen (O(ad)) and hydroxyl of LaFeO(3). The study points out a potential way to develop new and more active perovskite-type catalysts for the HMX thermal decomposition.

Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit

DOE PAGES

Das, Amit; Gerlits, Oksana O.; Heller, William T.; ...

2015-06-19

To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca 2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex,more » the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca 2+ cations with Mg 2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. As a result, the present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.« less

Energy Capture and Use in Plants and Bacteria. Final Technical Report

DOE R&D Accomplishments Database

Boyer, P. D.

1993-12-31

The project has centered on elucidation of the mechanism of ATP synthase. The metabolic importance of ATP and the complexity of the ATP synthase have made the problem particularly important and challenging. The development of the binding change mechanism depended upon our recognition of features that were novel in bioenergetics and indeed to the field of enzymology. One important feature of mechanism is that the principal way that energy input from transmembrane proton movement is coupled to ATP formation is to drive conformational changes that cause the release of ATP readily formed and tightly bound at a catalytic site. Another is that three equivalent catalytic sites on the enzyme show strong catalytic cooperativity as they proceed sequentially through different conformations. A more speculative features is that this cooperativity and energy coupling involve a rotational movement of minor subunits relative to the catalytic subunits. During this period these studies have extended and clarified aspects of the synthase mechanism. During assessments of interactions of Mg{sup 2+} and ADP with the synthase we recognized unexpectedly that whether ADP and P{sub i}, or their complexes with Mg{sup 2+} served as substrates for ATP formation by photophosphorylation was not known. Our studies showed that MgADP and free P{sub i} act as substrates.

The protein phosphatase 2A catalytic subunit StPP2Ac2b acts as a positive regulator of tuberization induction in Solanum tuberosum L.

PubMed

Muñiz García, María Noelia; Muro, María Catalina; Mazzocchi, Luciana Carla; País, Silvia Marina; Stritzler, Margarita; Schlesinger, Mariana; Capiati, Daniela Andrea

2017-02-01

This study provides the first genetic evidence for the role of PP2A in tuberization, demonstrating that the catalytic subunit StPP2Ac2b positively modulates tuber induction, and that its function is related to the regulation of gibberellic acid metabolism. The results contribute to a better understanding of the molecular mechanism controlling tuberization induction, which remains largely unknown. The serine/threonine protein phosphatases type 2A (PP2A) are implicated in several physiological processes in plants, playing important roles in hormone responses. In cultivated potato (Solanum tuberosum), six PP2A catalytic subunits (StPP2Ac) were identified. The PP2Ac of the subfamily I (StPP2Ac1, 2a and 2b) were suggested to be involved in the tuberization signaling in leaves, where the environmental and hormonal signals are perceived and integrated. The aim of this study was to investigate the role of PP2A in the tuberization induction in stolons. We selected one of the catalytic subunits of the subfamily I, StPP2Ac2b, to develop transgenic plants overexpressing this gene (StPP2Ac2b-OE). Stolons from StPP2Ac2b-OE plants show higher tuber induction rates in vitro, as compared to wild type stolons, with no differences in the number of tubers obtained at the end of the process. This effect is accompanied by higher expression levels of the gibberellic acid (GA) catabolic enzyme StGA2ox1. GA up-regulates StPP2Ac2b expression in stolons, possibly as part of the feedback system by which the hormone regulates its own level. Sucrose, a tuber-promoting factor in vitro, increases StPP2Ac2b expression. We conclude that StPP2Ac2b acts in stolons as a positive regulator tuber induction, integrating different tuberization-related signals mainly though the modulation of GA metabolism.

Expression of A-type K channel alpha subunits Kv 4.2 and Kv 4.3 in rat spinal lamina II excitatory interneurons and colocalization with pain-modulating molecules.

PubMed

Huang, Hsin-Yi; Cheng, Jen-Kun; Shih, Yang-Hsin; Chen, Pei-Hsuan; Wang, Chin-Lin; Tsaur, Meei-Ling

2005-09-01

Voltage-gated K(+) channel alpha subunits Kv 4.2 and Kv 4.3 are the major contributors of somatodendritic A-type K(+) currents in many CNS neurons. A recent hypothesis suggests that Kv 4 subunits may be involved in pain modulation in dorsal horn neurons. However, whether Kv 4 subunits are expressed in dorsal horn neurons remains unknown. Using immunohistochemistry, we found that Kv 4.2 and Kv 4.3 immunoreactivity was concentrated in the superficial dorsal horn, mainly in lamina II. Both Kv 4.2 and Kv 4.3 appeared on many rostrocaudally orientated dendrites, whereas Kv 4.3 could be also detected from certain neuronal somata. Kv 4.3(+) neurons were a subset of excitatory inerneurons with calretinin(+)/calbindin(-)/PKCgamma(-) markers, and a fraction of them expressed micro-opioid receptors. Kv 4.3(+) neurons also expressed ERK 2 and mGluR 5, which are molecules related to the induction of central sensitization, a mechanism mediating nociceptive plasticity. Together with the expression of Kv 4.3 in VR 1(+) DRG neurons, our data suggest that Kv C4 subunits could be involved in pain modulation.

Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

PubMed

Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

2004-01-01

AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antonacci, R.; Colombo, I.; Volta, M.

The electron-transfer flavoprotein (ETF), located in the mitochondrial matrix, is a nuclear-encoded enzyme delivering to the respiratory chain electrons by straight-chain acyl-CoA dehydrogenases and other dehydrogenases. ETF is composed of a 35-kDa [alpha]-subunit that is cleaved to a 32-kDa protein during mitochondrial import (ETFA) and a [beta]-subunit that reaches the mitochondrion unmodified (ETFB). The cDNA encoding both these subunits has been cloned and sequenced. 14 refs., 1 fig.

Teaching Glycoproteins with a Classical Paper: Knowledge and Methods in the Course of an Exciting Discovery--The story of Discovering HK-ATPase [Beta]-Subunit

ERIC Educational Resources Information Center

Zhu, Lixin

2008-01-01

To integrate research into the teaching of glycoproteins, the story of discovering hydrogen-potassium ATPase (HK-ATPase) [beta] subunit is presented in a way covering all the important teaching points. The interaction between the HK-ATPase [alpha] subunit and a glycoprotein of 60-80 kDa was demonstrated to support the existence of the [beta]…

Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matzuk, M.M.; Krieger, M.; Corless, C.L.

1987-09-01

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common ..cap alpha.. subunit but differ in their hormone-specific ..beta..-subunits. The ..beta.. subunit of hCG (hCG..beta..) is unique among the ..beta.. subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either hCG..beta.. gene alone or together with the hCG..cap alpha.. gene were transfected into a mutant Chinese hamster ovary cell line, 1d1D, which exhibits a reversible defect in O-glycosylation. The results revealmore » that hCG..beta.. can be secreted normally in the absence of its O-linked oligosaccharides. hCG..beta.. devoid of O-linked carbohydrate can also combine efficiently with hCG..cap alpha.. and be secreted as an intact dimer. The authors conclude that in Chinese hamster ovary cells, the hCG..beta.. O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG.« less

Alpha-momorcharin: a ribosome-inactivating protein from Momordica charantia, possessing DNA cleavage properties.

PubMed

Wang, Shuzhen; Zheng, Yinzhen; Yan, Junjie; Zhu, Zhixuan; Wu, Zhihua; Ding, Yi

2013-11-01

Ribosome-inactivating proteins (RIPs) function to inhibit protein synthesis through the removal of specific adenine residues from eukaryotic ribosomal RNA and rending the 60S subunit unable to bind elongation factor 2. They have received much attention in biological and biomedical research due to their unique activities toward tumor cells, as well as the important roles in plant defense. Alpha-momorcharin (α-MC), a member of the type I family of RIPs, is rich in the seeds of Momordica charantia L. Previous studies demonstrated that α-MC is an effective antifungal and antibacterial protein. In this study, a detailed analysis of the DNase-like activity of α-MC was conducted. Results showed that the DNase-like activity toward plasmid DNA was time-dependent, temperature-related, and pH-stable. Moreover, a requirement for divalent metal ions in the catalytic domain of α-MC was confirmed. Additionally, Tyr(93) was found to be a critical residue for the DNase-like activity, while Tyr(134), Glu(183), Arg(186), and Trp(215) were activity-related residues. This study on the chemico-physical properties and mechanism of action of α-MC will improve its utilization in scientific research, as well as its potential industrial uses. These results may also assist in the characterization and elucidation of the DNase-like enzymatic properties of other RIPs.

Differential expression of extracellular matrix molecules and the alpha 6-integrins in the normal and neoplastic prostate.

PubMed Central

Knox, J. D.; Cress, A. E.; Clark, V.; Manriquez, L.; Affinito, K. S.; Dalkin, B. L.; Nagle, R. B.

1994-01-01

The epithelial basal lamina composition and integrin expression profile of normal and neoplastic human prostate was characterized using immunohistochemical analysis of frozen samples. The major components of the basal lamina surrounding normal acini were laminin, type IV collagen, entactin, and type VII collagen with variable amounts of tenascin. The basal lamina of neoplastic acini had a similar composition, except for the loss of type VII collagen, which was observed in all grades of carcinoma. The basal cells of the normal prostate express the alpha 6-, beta 1-, and beta 4-integrin subunits, suggesting that both the alpha 6 beta 1- and alpha 6 beta 4-integrin complexes are formed. In prostate carcinoma there is a complete loss of beta 4 expression and the alpha 6- and beta 1-integrin subunits, which are restricted to the basal and basal lateral surfaces of basal cells, are distributed diffusely throughout the cytoplasmic membrane. The differential expression of type VII collagen and beta 4 are discussed in relationship to their possible role in tumor progression. Images Figure 1 Figure 2 Figure 3 PMID:8030747

The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

DOE PAGES

Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; ...

2014-08-11

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R 2:C 2), with a regulatory subunit homodimer (R 2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the typemore » IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.« less

The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

NASA Technical Reports Server (NTRS)

Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

1999-01-01

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

PubMed

Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

2014-10-10

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Subunit rotation of ATP synthase embedded in membranes: a or β subunit rotation relative to the c subunit ring

PubMed Central

Nishio, Kazuaki; Iwamoto-Kihara, Atsuko; Yamamoto, Akitsugu; Wada, Yoh; Futai, Masamitsu

2002-01-01

ATP synthase FoF1 (α3β3γδɛab2c10–14) couples an electrochemical proton gradient and a chemical reaction through the rotation of its subunit assembly. In this study, we engineered FoF1 to examine the rotation of the catalytic F1 β or membrane sector Fo a subunit when the Fo c subunit ring was immobilized; a biotin-tag was introduced onto the β or a subunit, and a His-tag onto the c subunit ring. Membrane fragments were obtained from Escherichia coli cells carrying the recombinant plasmid for the engineered FoF1 and were immobilized on a glass surface. An actin filament connected to the β or a subunit rotated counterclockwise on the addition of ATP, and generated essentially the same torque as one connected to the c ring of FoF1 immobilized through a His-tag linked to the α or β subunit. These results established that the γɛc10–14 and α3β3δab2 complexes are mechanical units of the membrane-embedded enzyme involved in rotational catalysis. PMID:12357031

Targeted deletion of the GABRA2 gene encoding alpha2-subunits of GABA(A) receptors facilitates performance of a conditioned emotional response, and abolishes anxiolytic effects of benzodiazepines and barbiturates.

PubMed

Dixon, C I; Rosahl, T W; Stephens, D N

2008-07-01

Mice with point-mutated alpha2 GABA(A) receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in the conditioned emotional response (CER) test, but show normal anxiolytic effects of a barbiturate. We investigated the consequence of deleting the alpha2-subunit on acquisition of the CER with increasing intensity of footshock, and on the anxiolytic efficacy of a benzodiazepine, diazepam, and a barbiturate, pentobarbital. alpha2 knockout (KO) and wildtype (WT) mice were trained in a conditioned emotional response (CER) task, in which lever pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a compound light/tone conditioned stimulus (CS+) that predicted footshock. The ability of diazepam and of pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. There were no differences between the genotypes in shock sensitivity, as assessed by their flinch responses to increasing levels of shock. However, alpha2 KO mice showed a greater suppression of lever pressing than WT littermates in the presence of a compound cue signalling footshock. Diazepam (0, 0.5, 1 and 2 mg/kg) induced a dose-dependent anxiolytic-like effect in WT mice but no such effect was seen in KO mice. Similarly, although pentobarbital (20 mg/kg) reduced the ability of the CS+ to reduce lever pressing rates in WT mice, this effect was not seen in the KO. These findings suggest that alpha2-containing GABA(A) receptors mediate the anxiolytic effects of barbiturates, as well as benzodiazepines, and that they may be involved in neuronal circuits underlying conditioned anxiety.

Asymmetry in the F1-ATPase and Its Implications for the Rotational Cycle

PubMed Central

Sun, Sean X.; Wang, Hongyun; Oster, George

2004-01-01

ATP synthase uses a rotary mechanism to carry out its cellular function of manufacturing ATP. The centralγ-shaft rotates inside a hexameric cylinder composed of alternating α- and β-subunits. When operating in the hydrolysis direction under high frictional loads and low ATP concentrations, a coordinated mechanochemical cycle in the three catalytic sites of the β-subunits rotates the γ-shaft in three 120° steps. At low frictional loads, the 120° steps alternate with three ATP-independent substeps separated by ∼30°. We present a quantitative model that accounts for these substeps and show that the observed pauses are due to 1), the asymmetry of the F1 hexamer that produces a propeller-like motion of the power-stroke and 2), the relatively tight binding of ADP to the catalytic sites. PMID:14990467

Characterization of the Catalytic Subunits of the RNA Exosome-like Complex in Plasmodium falciparum.

PubMed

Jiang, Ning; Yu, Shengchao; Yang, Na; Feng, Ying; Sang, Xiaoyu; Wang, Yao; Wahlgren, Mats; Chen, Qijun

2018-04-17

The eukaryotic ribonucleic acid (RNA) exosome is a versatile multiribonuclease complex that mediates the processing, surveillance, and degradation of virtually all classes of RNA in both the nucleus and cytoplasm. The complex, composed of 10 to 11 subunits, has been widely described in many organisms. Bioinformatic analyses revealed that there may be also an exosome-like complex in Plasmodium falciparum, a parasite of great importance in public health, with eight predicted subunits having high sequence similarity to their counterparts in yeast and human. In this work, the putative RNA catalytic components, designated as PfRrp4, PfRrp41, PfDis3, and PfRrp6, were identified and systematically analyzed. Quantitative polymerase chain reaction (QPCR) analyses suggested that all of them were transcribed steadily throughout the asexual stage. The expression of these proteins was determined by Western blot, and their localization narrowed to the cytoplasm of the parasite by indirect immunofluorescence. The recombinant proteins of PfRrp41, PfDis3, and PfRrp6 exhibited catalytic activity for single-stranded RNA (ssRNA), whereas PfRrp4 showed no processing activity of both ssRNA and dsRNA. The identification of these putative components of the RNA exosome complex opens up new perspectives for a deep understanding of RNA metabolism in the malarial parasite P. falciparum. © 2018 The Authors Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.

Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling.

PubMed Central

Janssens, V; Goris, J

2001-01-01

Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated. Regulation is accomplished mainly by members of a family of regulatory subunits, which determine the substrate specificity, (sub)cellular localization and catalytic activity of the PP2A holoenzymes. Moreover, the catalytic subunit is subject to two types of post-translational modification, phosphorylation and methylation, which are also thought to be important regulatory devices. The regulatory ability of PTPA (PTPase activator), originally identified as a protein stimulating the phosphotyrosine phosphatase activity of PP2A, will also be discussed, alongside the other regulatory inputs. The use of specific PP2A inhibitors and molecular genetics in yeast, Drosophila and mice has revealed roles for PP2A in cell cycle regulation, cell morphology and development. PP2A also plays a prominent role in the regulation of specific signal transduction cascades, as witnessed by its presence in a number of macromolecular signalling modules, where it is often found in association with other phosphatases and kinases. Additionally, PP2A interacts with a substantial number of other cellular and viral proteins, which are PP2A substrates, target PP2A to different subcellular compartments or affect enzyme activity. Finally, the de-regulation of PP2A in some specific pathologies will be touched upon. PMID:11171037

Both α and β Subunits of Human Choriogonadotropin Photoaffinity Label the Hormone Receptor

NASA Astrophysics Data System (ADS)

Ji, Inhae; Ji, Tae H.

1981-09-01

It has been shown that a photoactivable derivative of human choriogonadotropin (hCG) labels the lutropin receptor on porcine granulosa cells [Ji, I. & Ji, T. H. (1980) Proc. Natl. Acad. Sci. USA 77, 7167-7170]. In an attempt to identify which of the hCG subunits labeled the receptor, three sets of different hCG derivatives were prepared. In the first set, hCG was coupled to the N-hydroxysuccinimide ester of 4-azidobenzoylglycine and radioiodinated. In the second set, only one of the subunits was radioiodinated, but both subunits were allowed to react with the reagent. In the third set, both the reagent and [125I]iodine were coupled to only one of the subunits. The binding activity of each hormone derivative was comparable to that of 125I-labeled hCG. After binding of these hormone derivatives to the granulosa cell surface, they were photolyzed. After solubilization, autoradiographs of sodium dodecyl sulfate/polyacrylamide gels of each sample revealed a number of labeled bands; the hCG derivatives containing 125I-labeled alpha subunit produced four bands (molecular weights 120,000 +/- 6,000, 96,000 +/- 5,000, 76,000 +/- 4,000, and 73,000 +/- 4,000) and those containing 125I-labeled beta subunit produced three bands (molecular weights 106,000 +/- 6,000, 88,000 +/- 5,000, and 83,000 +/- 4,000). Results were the same when the hormone-receptor complexes were solubilized in 0.5% Triton X-100 and then photolyzed or when the hormone was derivatized with a family of reagents having arms of various lengths. We conclude that both the alpha subunit and the beta subunit of hCG photoaffinity labeled certain membrane polypeptides and that these polypeptides are related to the hormone receptor.

Effect of alternative glycosylation on insulin receptor processing.

PubMed

Hwang, J B; Frost, S C

1999-08-06

The mature insulin receptor is a cell surface heterotetrameric glycoprotein composed of two alpha- and two beta-subunits. In 3T3-L1 adipocytes as in other cell types, the receptor is synthesized as a single polypeptide consisting of uncleaved alpha- and beta-subunits, migrating as a 190-kDa glycoprotein. To examine the importance of N-linked glycosylation on insulin receptor processing, we have used glucose deprivation as a tool to alter protein glycosylation. Western blot analysis shows that glucose deprivation led to a time-dependent accumulation of an alternative proreceptor of 170 kDa in a subcellular fraction consistent with endoplasmic reticulum localization. Co-precipitation assays provide evidence that the alternative proreceptor bound GRP78, an endoplasmic reticulum molecular chaperone. N-Glycosidase F treatment shows that the alternative proreceptor contained N-linked oligosaccharides. Yet, endoglycosidase H insensitivity indicates an aberrant oligosaccharide structure. Using pulse-chase methodology, we show that the synthetic rate was similar between the normal and alternative proreceptor. However, the normal proreceptor was processed into alpha- and beta-subunits (t((1)/(2)) = 1.3 +/- 0.6 h), while the alternative proreceptor was degraded (t((1)/(2)) = 5.1 +/- 0.6 h). Upon refeeding cells that were initially deprived of glucose, the alternative proreceptor was processed to a higher molecular weight form and gained sensitivity to endoglycosidase H. This "intermediate" form of the proreceptor was also degraded, although a small fraction escaped degradation, resulting in cleavage to the alpha- and beta-subunits. These data provide evidence for the first time that glucose deprivation leads to the accumulation of an alternative proreceptor, which can be post-translationally glycosylated with the readdition of glucose inducing both accelerated degradation and maturation.

KChIPs and Kv4 alpha subunits as integral components of A-type potassium channels in mammalian brain.

PubMed

Rhodes, Kenneth J; Carroll, Karen I; Sung, M Amy; Doliveira, Lisa C; Monaghan, Michael M; Burke, Sharon L; Strassle, Brian W; Buchwalder, Lynn; Menegola, Milena; Cao, Jie; An, W Frank; Trimmer, James S

2004-09-08

Voltage-gated potassium (Kv) channels from the Kv4, or Shal-related, gene family underlie a major component of the A-type potassium current in mammalian central neurons. We recently identified a family of calcium-binding proteins, termed KChIPs (Kv channel interacting proteins), that bind to the cytoplasmic N termini of Kv4 family alpha subunits and modulate their surface density, inactivation kinetics, and rate of recovery from inactivation (An et al., 2000). Here, we used single and double-label immunohistochemistry, together with circumscribed lesions and coimmunoprecipitation analyses, to examine the regional and subcellular distribution of KChIPs1-4 and Kv4 family alpha subunits in adult rat brain. Immunohistochemical staining using KChIP-specific monoclonal antibodies revealed that the KChIP polypeptides are concentrated in neuronal somata and dendrites where their cellular and subcellular distribution overlaps, in an isoform-specific manner, with that of Kv4.2 and Kv4.3. For example, immunoreactivity for KChIP1 and Kv4.3 is concentrated in the somata and dendrites of hippocampal, striatal, and neocortical interneurons. Immunoreactivity for KChIP2, KChIP4, and Kv4.2 is concentrated in the apical and basal dendrites of hippocampal and neocortical pyramidal cells. Double-label immunofluorescence labeling revealed that throughout the forebrain, KChIP2 and KChIP4 are frequently colocalized with Kv4.2, whereas in cortical, hippocampal, and striatal interneurons, KChIP1 is frequently colocalized with Kv4.3. Coimmunoprecipitation analyses confirmed that all KChIPs coassociate with Kv4 alpha subunits in brain membranes, indicating that KChIPs 1-4 are integral components of native A-type Kv channel complexes and are likely to play a major role as modulators of somatodendritic excitability.

Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miles, K.; Anthony, D.T.; Rubin, L.L.

1987-09-01

The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with (/sup 32/P)orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the ..beta.. and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the ..cap alpha.. subunit. The effect of forskolin was dose dependent with a half-maximalmore » response at 8 ..mu..M in the presence of 35 ..mu..M Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of ..cap alpha.. subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes.« less

Mining the Immune Cell Proteome to Identify Ovarian Cancer-Specific Biomarkers

DTIC Science & Technology

2013-11-01

transcription 4 (STAT4) Ras-related C3 botulinum toxin substrate 3 (RAC3) Serine/ threonine -protein phosphatase 2A catalytic subunit beta isoform (PP2AB...Mitogen-activated protein kinase 14 (MK14) Wnt signaling pathway (6) Beta-arrestin-1 (ARRB1) Serine/ threonine -protein phosphatase 2A catalytic...carried the diagnosis of chronic hypertension, diabetes , anti-phospholipid lipid antibody syndrome, or systemic lupus erythematous. Subjects were also

RUNX1 regulates phosphoinositide 3-kinase/AKT pathway: role in chemotherapy sensitivity in acute megakaryocytic leukemia.

PubMed

Edwards, Holly; Xie, Chengzhi; LaFiura, Katherine M; Dombkowski, Alan A; Buck, Steven A; Boerner, Julie L; Taub, Jeffrey W; Matherly, Larry H; Ge, Yubin

2009-09-24

RUNX1 (AML1) encodes the core binding factor alpha subunit of a heterodimeric transcription factor complex which plays critical roles in normal hematopoiesis. Translocations or down-regulation of RUNX1 have been linked to favorable clinical outcomes in acute leukemias, suggesting that RUNX1 may also play critical roles in chemotherapy responses in acute leukemias; however, the molecular mechanisms remain unclear. The median level of RUNX1b transcripts in Down syndrome (DS) children with acute megakaryocytic leukemia (AMkL) were 4.4-fold (P < .001) lower than that in non-DS AMkL cases. Short hairpin RNA knockdown of RUNX1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivity to cytosine arabinoside, accompanied by significantly decreased expression of PIK3CD, which encodes the delta catalytic subunit of the survival kinase, phosphoinositide 3 (PI3)-kinase. Transcriptional regulation of PIK3CD by RUNX1 was further confirmed by chromatin immunoprecipitation and promoter reporter gene assays. Further, a PI3-kinase inhibitor, LY294002, and cytosine arabinoside synergized in antileukemia effects on Meg-01 and primary pediatric AMkL cells. Our results suggest that RUNX1 may play a critical role in chemotherapy response in AMkL by regulating the PI3-kinase/Akt pathway. Thus, the treatment of AMkL may be improved by integrating PI3-kinase or Akt inhibitors into the chemotherapy of this disease.

The 60 kDa heat shock proteins in the hyperthermophilic archaeon Sulfolobus shibatae.

PubMed

Kagawa, H K; Osipiuk, J; Maltsev, N; Overbeek, R; Quaite-Randall, E; Joachimiak, A; Trent, J D

1995-11-10

One of the most abundant proteins in the hyperthermophilic archaeon Sulfolobus shibatae is the 59 kDa heat shock protein (TF55) that is believed to form a homo-oligomeric double ring complex structurally similar to the bacterial chaperonins. We discovered a second protein subunit in the S. shibatae ring complex (referred to as alpha) that is stoichiometric with TF55 (renamed beta). The gene and flanking regions of alpha were cloned and sequenced and its inferred amino acid sequence has 54.4% identity and 74.4% similarity to beta. Transcription start sites for both alpha and beta were mapped and three potential transcription regulatory regions were identified. Northern analyses of cultures shifted from normal growth temperatures (70 to 75 degrees C) to heat shock temperatures (85 to 90 degrees C) indicated that the levels of alpha and beta mRNAs increased during heat shock, but at all temperatures their relative proportions remained constant. Monitoring protein synthesis by autoradiography of total proteins from cultures pulse labeled with L(-)[35S]methionine at normal and heat shock temperatures indicated significant increases in alpha and beta synthesis during heat shock. Under extreme heat shock conditions (> or = 90 degrees C) alpha and beta appeared to be the only two proteins synthesized. The purified alpha and beta subunits combined to form high molecular mass complexes with similar mobilities on native polyacrylamide gels to the complexes isolated directly from cells. Equal proportions of the two subunits gave the greatest yield of the complex, which we refer to as a "rosettasome". It is argued that the rosettasome consists of two homo-oligomeric rings; one of alpha and the other of beta. Polyclonal antibodies against alpha and beta from S. shibatae cross-reacted with proteins of similar molecular mass in 10 out of the 17 archaeal species tested, suggesting that the two rosettasome proteins are highly conserved among the archaea. The archaeal sequences were aligned with bacterial and eukaryotic chaperonins to generate a phylogenetic tree. The tree reveals the close relationship between the archaeal rosettasomes and the eukaryotic TCP1 protein family and the distant relationship to the bacterial GroEL/HSP60 proteins.

Identification of Telomerase Components and Telomerase Regulating Factors in Yeast

DTIC Science & Technology

1998-07-01

subunit of telomerase in S. cerevisiae is encoded by TLCJ (7). Recently , through sequence comparison with the telomerase catalytic 6 subunit from Euplotes...length maintenance has been unclear, although very recent data has shown that Ku80p can be found specifically associated with telomeric DNA in vivo...chromatin structure. It has been recently observed that loss of either YKU80 or HDF1 results in altered telomere end structure, such that there appears to

Regulation of PSMB5 Protein and β Subunits of Mammalian Proteasome by Constitutively Activated Signal Transducer and Activator of Transcription 3 (STAT3)

PubMed Central

Vangala, Janakiram Reddy; Dudem, Srikanth; Jain, Nishant; Kalivendi, Shasi V.

2014-01-01

The ubiquitin-proteasome system facilitates the degradation of ubiquitin-tagged proteins and performs a regulatory role in cells. Elevated proteasome activity and subunit expression are found in several cancers. However, the inherent molecular mechanisms responsible for increased proteasome function in cancers remain unclear despite the well investigated and defined role of the mammalian proteasome. This study was initiated to elucidate the mechanisms involved in the regulation of β subunits of the mammalian proteasome. Suppression of STAT3 tyrosine phosphorylation coordinately decreased the mRNA and protein levels of the β subunits of the 20 S core complex in DU145 cells. Notably, PSMB5, a molecular target of bortezomib, was shown to be a target of STAT3. Knockdown of STAT3 decreased PSMB5 protein. Inhibition of phospho-STAT3 substantially reduced PSMB5 protein levels in cells expressing constitutively active-STAT3. Accumulation of activated STAT3 resulted in the induction of PSMB5 promoter and protein levels. In addition, a direct correlation was observed between the endogenous levels of PSMB5 and constitutively active STAT3. PSMB5 and STAT3 protein levels remained unaltered following the inhibition of proteasome activity. The EGF-induced concerted increase of β subunits was blocked by inhibition of the EGF receptor or STAT3 but not by the PI3K/AKT or MEK/ERK pathways. Decreased proteasome activities were due to reduced protein levels of catalytic subunits of the proteasome in STAT3-inhibited cells. Combined treatments with bortezomib and inhibitor of STAT3 abrogated proteasome activity and enhanced cellular apoptosis. Overall, we demonstrate that aberrant activation of STAT3 regulates the expression of β subunits, in particular PSMB5, and the catalytic activity of the proteasome. PMID:24627483

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

PubMed Central

Faurobert, E; Otto-Bruc, A; Chardin, P; Chabre, M

1993-01-01

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding. Images PMID:8223434

ε Subunit of Bacillus subtilis F1-ATPase Relieves MgADP Inhibition

PubMed Central

Mizumoto, Junya; Kikuchi, Yuka; Nakanishi, Yo-Hei; Mouri, Naoto; Cai, Anrong; Ohta, Tokushiro; Haruyama, Takamitsu; Kato-Yamada, Yasuyuki

2013-01-01

MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF1. We conclude that the ε subunit relieves BF1 from MgADP inhibition. PMID:23967352

Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

PubMed Central

Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R

1992-01-01

The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568

Effect of metal ions on the activity of casein kinase II from Xenopus laevis.

PubMed

Gatica, M; Hinrichs, M V; Jedlicki, A; Allende, C C; Allende, J E

1993-01-04

Casein kinase II purified from the nuclei of Xenopus laevis oocytes as well as the recombinant alpha and beta subunits of the X. laevis CKII, produced in E. coli from the cloned cDNA genes, were tested with different divalent metal ions. The enzyme from both sources was active with either Mg2+, Mn2+, or Co2+. Optimal concentrations were 7-10 mM for Mg2+, 0.5-0.7 mM for Mn2+ and 1-2 mM for Co2+. In the presence of Mn2+ or Co2+ the enzyme used GTP more efficiently than ATP as a phosphate donor while the reverse was true in the presence of Mg2+. The apparent Km values for both nucleotide triphosphates were greatly decreased in the presence of Mn2+ as compared with Mg2+. Addition of Zn2+ (above 150 microM) to an assay containing the optimal Mg2+ ion concentration caused strong inhibition of both holoenzyme and alpha subunit. Inhibition of the holoenzyme by 400 microM Ni2+ could be reversed by high concentrations of Mg2+ but no reversal of this inhibition was observed with the alpha subunit.

Modulation of A-type potassium channels by a family of calcium sensors.

PubMed

An, W F; Bowlby, M R; Betty, M; Cao, J; Ling, H P; Mendoza, G; Hinson, J W; Mattsson, K I; Strassle, B W; Trimmer, J S; Rhodes, K J

2000-02-03

In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.

Finding molecular dioxygen tunnels in homoprotocatechuate 2,3-dioxygenase: implications for different reactivity of identical subunits.

PubMed

Xu, Liang; Zhao, Weijie; Wang, Xicheng

2010-01-01

Extradiol dioxygenases facilitate microbial aerobic degradation of catechol and its derivatives by activating molecular dioxygen and incorporating both oxygen atoms into their substrates. Experimental and theoretical studies have focused on the mechanism of the reaction at the active site. However, whether the catalytic rate is limited by O(2) access to the active site has not yet been explored. Here, we choose a recently solved X-ray structure of homoprotocatechuate 2,3-dioxygenase as a typical example to determine potential pathways for O(2) migration from the solvent into the enzyme center. On the basis of the trajectories of two 10-ns molecular dynamics simulations, implicit ligand sampling was used to calculate the 3D free energy map for O(2) inside the protein. The energetically optimal routes for O(2) diffusion were identified for each subunit of the homotetrameric protein structure. The O(2) tunnels formed because of thermal fluctuations were also characterized by connecting elongated cavities inside the protein. By superimposing the favorable O(2) tunnels on to the free energy map, both energetically and geometrically preferred O(2) pathways were determined, as also were the amino acids that may be critical for O(2) passage along these paths. Our results demonstrate that identical subunits possess quite distinct O(2) tunnels. The order of O(2) affinity of these tunnels is generally consistent with the order of the catalytic rate of each subunit. As a consequence, the probability of finding the reaction product is highest in the subunit containing the highest O(2) affinity pathway.

N-Glycosylation of the alpha subunit does not influence trafficking or functional activity of the human organic solute transporter alpha/beta

PubMed Central

Soroka, Carol J; Xu, Shuhua; Mennone, Albert; Lam, Ping; Boyer, James L

2008-01-01

Background The organic solute transporter (OSTα-OSTβ) is a heteromeric transporter that is expressed on the basolateral membrane of epithelium in intestine, kidney, liver, testis and adrenal gland and facilitates efflux of bile acids and other steroid solutes. Both subunits are required for plasma membrane localization of the functional transporter but it is unclear how and where the subunits interact and whether glycosylation is required for functional activity. We sought to examine these questions for the human OSTα-OSTβ transporter using the human hepatoma cell line, HepG2, and COS7 cells transfected with constructs of human OSTα-FLAG and OSTβ-Myc. Results Tunicamycin treatment demonstrated that human OSTα is glycosylated. In COS7 cells Western blotting identified the unglycosylated form (~31 kD), the core precursor form (~35 kD), and the mature, complex glycoprotein (~40 kD). Immunofluorescence of both cells indicated that, in the presence of OSTβ, the alpha subunit could still be expressed on the plasma membrane after tunicamycin treatment. Furthermore, the functional uptake of 3H-estrone sulfate was unchanged in the absence of N-glycosylation. Co-immunoprecipitation indicates that the immature form of OSTα interact with OSTβ. However, immunoprecipitation of OSTβ using an anti-Myc antibody did not co-precipitate the mature, complex glycosylated form of OSTα, suggesting that the primary interaction occurs early in the biosynthetic pathway and may be transient. Conclusion In conclusion, human OSTα is a glycoprotein that requires interaction with OSTβ to reach the plasma membrane. However, glycosylation of OSTα is not necessary for interaction with the beta subunit or for membrane localization or function of the heteromeric transporter. PMID:18847488

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petroulakis, E.; Cao, Z.; Salo, T.

Mutations in the HEXA gene that encodes the {alpha}-subunit of the heterodimeric lysosomal enzyme {beta}-hexosaminidase A, or Hex A ({alpha}{beta}), cause G{sub M2} gangliosidosis, type 1. The infantile form (Tay-Sachs disease) results when there is no residual Hex A activity, while less severe and more variable clinical phenotypes result when residual Hex A activity is present. A non-Jewish male who presented with an acute psychotic episode at age 16 was diagnosed with a subacute encephalopathic form of G{sub M2} gangliosidosis. At age 19, chronic psychosis with intermittent acute exacerbations remains the most disabling symptom in this patient and his affectedmore » brother although both exhibit some ataxia and moderately severe dysarthria. We have found a 4 bp insertion (+TATC 1278) associated with infantile Tay-Sachs disease on one allele; no previously identified mutation was found on the second allele. SSCP analysis detected a shift in exon 13 and sequencing revealed a G1422C mutation in the second allele that results in a Trp474Cys substitution. The presence of the mutation was confirmed by the loss of HaeIII and ScrFI sites in exon 13 PCR products from the subjects and their father. The mutation was introduced into the {alpha}-subunit cDNA and Hex S ({alpha}{alpha}) and Hex A ({alpha}{beta}) were transiently expressed in monkey COS-7 cells. The Trp474Cys mutant protein had approximately 5% and 12% of wild-type Hex S and Hex A activity, respectively. Western blot analysis revealed a small amount of residual mature {alpha}-subunit and a normal level of precursor protein. We conclude that the Trp474Cys mutation is the cause of the Hex A deficiency associated with a subacute (juvenile-onset) phenotype in this patient. Like other mutations in exon 13 of HEXA, it appears to affect intracellular processing. Studies of the defect in intracellular processing are in progress.« less

The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPase alpha 3 subunit gene are required for brain expression in transgenic mice.

PubMed Central

Pathak, B G; Neumann, J C; Croyle, M L; Lingrel, J B

1994-01-01

The Na,K-ATPase is an integral plasma membrane protein consisting of alpha and beta subunits, each of which has discrete isoforms expressed in a tissue-specific manner. Of the three functional alpha isoform genes, the one encoding the alpha 3 isoform is the most tissue-restricted in its expression, being found primarily in the brain. To identify regions of the alpha 3 isoform gene that are involved in directing expression in the brain, a 1.6 kb 5'-flanking sequence was attached to a reporter gene, chloramphenicol acetyltransferase (CAT). The alpha 3-CAT chimeric gene construct was microinjected into fertilized mouse eggs, and transgenic mice were produced. Analysis of adult transgenic mice from different lines revealed that the transgene is expressed primarily in the brain. To further delineate regions that are needed for conferring expression in this tissue, systematic deletions of the 5'-flanking sequence of the alpha 3-CAT fusion constructs were made and analyzed, again using transgenic mice. The results from these analyses indicate that DNA sequences required for mediating brain-specific expression of the alpha 3 isoform gene are present within 210 bp upstream of the transcription initiation site. alpha 3-CAT promoter constructs containing scanning mutations in this region were also assayed in transgenic mice. These studies have identified both a functional neural-restrictive silencer element as well as a positively acting cis element. Images PMID:7984427

Three alpha-subunits of heterotrimeric G proteins and an adenylyl cyclase have distinct roles in fruiting body development in the homothallic fungus Sordaria macrospora.

PubMed

Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

2008-09-01

Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different alpha-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Deltagsa1, Deltagsa2, and Deltagsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Galpha-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Deltagsa1Deltagsa2 and Deltagsa1Deltagsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Galpha-subunits, two recently generated Deltapre strains were crossed with all Deltagsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three DeltagsaDeltasac1 double mutants and one Deltagsa2Deltagsa3Deltasac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Galpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

Crystal Structure of the MACPF Domain of Human Complement Protein C8[alpha] in Complex with the C8[gamma] Subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slade, Daniel J.; Lovelace, Leslie L.; Chruszcz, Maksymilian

2010-03-04

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that assemble on bacterial membranes to form a porelike structure referred to as the 'membrane attack complex' (MAC). C8 contains three genetically distinct subunits (C8{alpha}, C8{beta}, C8{gamma}) arranged as a disulfide-linked C8{alpha}-{gamma} dimer that is noncovalently associated with C8{beta}. C6, C7 C8{alpha}, C8{beta}, and C9 are homologous. All contain N- and C-terminal modules and an intervening 40-kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. The C8{gamma} subunit is unrelated and belongs to the lipocalin family of proteins that display a {beta}-barrel fold andmore » generally bind small, hydrophobic ligands. Several hundred proteins with MACPF domains have been identified based on sequence similarity; however, the structure and function of most are unknown. Crystal structures of the secreted bacterial protein Plu-MACPF and the human C8{alpha} MACPF domain were recently reported and both display a fold similar to those of the bacterial pore-forming cholesterol-dependent cytolysins (CDCs). In the present study, we determined the crystal structure of the human C8{alpha} MACPF domain disulfide-linked to C8{gamma} ({alpha}MACPF-{gamma}) at 2.15 {angstrom} resolution. The {alpha}MACPF portion has the predicted CDC-like fold and shows two regions of interaction with C8{gamma}. One is in a previously characterized 19-residue insertion (indel) in C8{alpha} and fills the entrance to the putative C8{gamma} ligand-binding site. The second is a hydrophobic pocket that makes contact with residues on the side of the C8{gamma} {beta}-barrel. The latter interaction induces conformational changes in {alpha}MACPF that are likely important for C8 function. Also observed is structural conservation of the MACPF signature motif Y/W-G-T/S-H-F/Y-X{sub 6}-G-G in {alpha}MACPF and Plu-MACPF, and conservation of several key glycine residues known to be important for refolding and pore formation by CDCs.« less

Function and selectivity of bromodomains in anchoring chromatin-modifying complexes to promoter nucleosomes.

PubMed

Hassan, Ahmed H; Prochasson, Philippe; Neely, Kristen E; Galasinski, Scott C; Chandy, Mark; Carrozza, Michael J; Workman, Jerry L

2002-11-01

The functions of the SAGA and SWI/SNF complexes are interrelated and can form stable "epigenetic marks" on promoters in vivo. Here we show that stable promoter occupancy by SWI/SNF and SAGA in the absence of transcription activators requires the bromodomains of the Swi2/Snf2 and Gcn5 subunits, respectively, and nucleosome acetylation. This acetylation can be brought about by either the SAGA or NuA4 HAT complexes. The bromodomain in the Spt7 subunit of SAGA is dispensable for this activity but will anchor SAGA if it is swapped into Gcn5, indicating that specificity of bromodomain function is determined in part by the subunit it occupies. Thus, bromodomains within the catalytic subunits of SAGA and SWI/SNF anchor these complexes to acetylated promoter nucleosomes.

High frequency of coexistent mutations of PIK3CA and PTEN genes in endometrial carcinoma.

PubMed

Oda, Katsutoshi; Stokoe, David; Taketani, Yuji; McCormick, Frank

2005-12-01

The phosphatidylinositol 3'-kinase (PI3K) pathway is activated in many human cancers. In addition to inactivation of the PTEN tumor suppressor gene, mutations or amplifications of the catalytic subunit alpha of PI3K (PIK3CA) have been reported. However, the coexistence of mutations in these two genes seems exceedingly rare. As PTEN mutations occur at high frequency in endometrial carcinoma, we screened 66 primary endometrial carcinomas for mutations in the helical and catalytic domains of PIK3CA. We identified a total of 24 (36%) mutations in this gene and coexistence of PIK3CA/PTEN mutations at high frequency (26%). PIK3CA mutations were more common in tumors with PTEN mutations (17 of 37, 46%) compared with those without PTEN mutations (7 of 29, 24%). Array comparative genomic hybridization detected 3q24-qter amplification, which covers the PIK3CA gene (3q26.3), in one of nine tumors. Knocking down PTEN expression in the HEC-1B cell line, which possesses both K-Ras and PIK3CA mutations, further enhances phosphorylation of Akt (Ser473), indicating that double mutation of PIK3CA and PTEN has an additive effect on PI3K activation. Our data suggest that the PI3K pathway is extensively activated in endometrial carcinomas, and that combination of PIK3CA/PTEN alterations might play an important role in development of these tumors.

Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Kap; Pullalarevu, Sadhana; Surabian, Karen Talin

2010-03-12

Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg{sup 2+}-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phosphoglycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicelullar organisms that encounter high and variable energy turnover. GK from the marine worm Namalycastis sp. is heterodimeric, with two homologous polypeptide chains, {alpha} and {beta}, derived from a common pre-mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GK{beta} encodes a peptide that is different in sequence and is 16 amino acids longermore » than that encoded by the N-terminal exon of GK{alpha}. The crystal structures of recombinant GK{alpha}{beta} and GK{beta}{beta} from Namalycastis sp. were determined at 2.6 and 2.4 {angstrom} resolution, respectively. In addition, the structure of the GK{beta}{beta} was determined at 2.3 {angstrom} resolution in complex with a transition state analogue, Mg{sup 2+}-ADP-NO{sub 3}{sup -}-glycocyamine. Consistent with the sequence homology, the GK subunits adopt the same overall fold as that of other phosphagen kinases of known structure (the homodimeric creatine kinase (CK) and the monomeric arginine kinase (AK)). As with CK, the GK N-termini mediate the dimer interface. In both heterodimeric and homodimeric GK forms, the conformations of the two N-termini are asymmetric, and the asymmetry is different than that reported previously for the homodimeric CKs from several organisms. The entire polypeptide chains of GK{alpha}{beta} are structurally defined, and the longer N-terminus of the {beta} subunit is anchored at the dimer interface. In GK{beta}{beta} the 24 N-terminal residues of one subunit and 11 N-terminal residues of the second subunit are disordered. This observation is consistent with a proposal that the GK{alpha}{beta} amino acids involved in the interface formation were optimized once a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer interface (either solely {alpha} or solely {beta} chains) has been corrupted. In the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analogue, both subunits of GK undergo the same closure motion that clasps the transition state analogue, in contrast to the transition state analogue complexes of CK, where the corresponding transition state analogue occupies only one subunit, which undergoes domain closure. The active site environments of the GK, CK, and AK at the bound states reveal the structural determinants of substrate specificity. Despite the equivalent binding in both active sites of the GK dimer, the conformational asymmetry of the N-termini is retained. Thus, the coupling between the structural asymmetry and negative cooperativity previously proposed for CK is not supported in the case of GK.« less

Functions and ATP-binding responses of the twelve histidine residues in the TF1-ATPase beta subunit.

PubMed

Tozawa, K; Yagi, H; Hisamatsu, K; Ozawa, K; Yoshida, M; Akutsu, H

2001-10-01

The C2 proton signals of all (twelve) histidine residues of the TF1 beta subunit in the 1H-NMR spectrum have been identified and assigned by means of pH change experiments and site-directed substitution of histidines by glutamines. pH and ligand titration experiments were carried out for these signals. Furthermore, the ATPase activity of the reconstituted alpha3beta3gamma complex was examined for the twelve mutant beta subunits. Two of three conserved histidines, namely, His-119 and 324, were found to be important for expression of the ATPase activity. The former fixes the N-terminal domain to the central domain. His-324 is involved in the formation of the interface essential for the alpha3beta3gamma complex assembly. The other conserved residue, His-363, showed a very low pK(a), suggesting that it is involved in the tertiary structure formation. On the binding of a nucleotide, only the signals of His-173, 179, 200, and 324 shifted. These histidines are located in the hinge region, and its proximity, of the beta subunit. This observation provided further support for the conformational change of the beta monomer from the open to the closed form on the binding of a nucleotide proposed by us [Yagi et al. (1999) Biophys. J. 77, 2175-2183]. This conformational change should be one of the essential driving forces in the rotation of the alpha3beta3gamma complex.

Binding symmetry of extracellular divalent cations to conduction pore studied using tandem dimers of a CNG channel.

PubMed

Kwon, Ryuk-Jun; Ha, Tal Soo; Kim, Wonjae; Park, Chul-Seung

2002-11-08

Cyclic nucleotide-gated (CNG) channels are composed of the tetramer of alpha-subunit alone or alpha- and beta-subunits. The alpha-subunits of these channels have a conserved glutamate (Glu) residue within the pore-forming region and the residue determines the selectivity as well as the affinity for the extracellular divalent cations. Using the high-affinity mutant (E363D) of bovine retinal CNG channel in which the Glu at position 363 was replaced to Asp, we constructed tandem dimers and investigated the binding characteristics of divalent cations to the site. The gating and permeation characteristics of individual homomeric tandem dimers are indistinguishable to those of homo-tetramers formed by parental monomers. The heteromeric tandem dimers showed the binding affinity for Sr(2+) identical to the geometric mean of the affinities for two parent channels, indicating the energy additive and thus the simultaneous interaction. On the other hand, the binding affinity for Mg(2+) followed the harmonic mean of those parent channels indicating that Mg(2+) interacts more strongly with the subunit bearing Asp residue at the position. Thus the results strongly suggest that the Glu363 residues in the CNG channel pore be flexible enough to adapt different binding symmetries for different divalent cations. Moreover, the simultaneous interaction between the four Glu residues and Sr(2+) provides an important structural constraint to the CNG channel outer vestibule of unknown structure.

Substitution of histidine-137 by glutamine abolishes the catalytic activity of the ribosome-inactivating protein alpha-sarcin.

PubMed Central

Lacadena, J; Mancheño, J M; Martinez-Ruiz, A; Martínez del Pozo, A; Gasset, M; Oñaderra, M; Gavilanes, J G

1995-01-01

The alpha-sarcin cytotoxin is an extracellular fungal protein that inhibits protein biosynthesis by specifically cleaving one phosphodiester bond of the 28 S rRNA. The His137 residue of alpha-sarcin is suggested to be involved in the catalytic activity of this protein, based on the observed sequence similarity with some fungal ribonucleases. Replacement of this residue by Gln (H137Q mutant variant of alpha-sarcin) abolishes the ribonuclease activity of the protein. This has been demonstrated for an homogeneous preparation of the H137Q alpha-sarcin by measuring its effect against both intact rabbit ribosomes and the homopolymer poly(A). The conformation of H137Q alpha-sarcin is highly similar to that of the wild-type protein, which has been analysed by CD and fluorescence spectroscopy. Both H137Q and wild-type alpha-sarcin exhibit identical CD spectra in the peptide-bond region, indicating that no changes at the level of the secondary structure are produced upon mutation. Only minor differences are observed in both near-UV CD and fluorescence emission spectra in comparison to those of the wild-type protein. Moreover, H137Q alpha-sarcin interacts with phospholipid vesicles, promoting the same effects as the native cytotoxin. Therefore, we propose that His137 is part of the ribonucleolytic active site of the cytotoxin alpha-sarcin. Images Figure 4 PMID:7626023

Luminal Starch Substrate "Brake" on Maltase-Glucoamylase Activity Is Located within the Glucoamylase Subunit

USDA-ARS?s Scientific Manuscript database

The detailed mechanistic aspects for the final starch digestion process leading to effective alpha-glucogenesis by the 2 mucosal alpha-glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast v...

Mechanics of coupling proton movements to c-ring rotation in ATP synthase.

PubMed

Fillingame, Robert H; Angevine, Christine M; Dmitriev, Oleg Y

2003-11-27

F1F0 ATP synthases generate ATP by a rotary catalytic mechanism in which H+ transport is coupled to rotation of an oligomeric ring of c subunits extending through the membrane. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Subunit a of the F0 sector is thought to provide proton access channels to and from aspartyl-61. Here, we summarize new information on the structural organization of Escherichia coli subunit a and the mapping of aqueous-accessible residues in the second, fourth and fifth transmembrane helices (TMHs). Aqueous-accessible regions of these helices extend to both the cytoplasmic and periplasmic surface. We propose that aTMH4 rotates to alternately expose the periplasmic or cytoplasmic half-channels to aspartyl-61 of subunit c during the proton transport cycle. The concerted rotation of interacting helices in subunit a and subunit c is proposed to be the mechanical force driving rotation of the c-rotor, using a mechanism akin to meshed gears.

Structural Basis of PP2A Inhibition by Small t Antigen

PubMed Central

Cho, Uhn Soo; Morrone, Seamus; Sablina, Anna A; Arroyo, Jason D; Hahn, William C; Xu, Wenqing

2007-01-01

The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 Å resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3–7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST. PMID:17608567

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity.

PubMed

Ren, Huimiao; Bandyopadhyay, Sanjay; Allison, William S

2006-05-16

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.

Human DNA polymerase ε is phosphorylated at serine-1940 after DNA damage and interacts with the iron-sulfur complex chaperones CIAO1 and MMS19

PubMed Central

Moiseeva, Tatiana; Gamper, Armin M.; Hood, Brian; Conrads, Thomas P.; Bakkenist, Christopher J.

2016-01-01

We describe a dynamic phosphorylation on serine-1940 of the catalytic subunit of human Pol ε, POLE1, following DNA damage. We also describe novel interactions between POLE1 and the iron-sulfur cluster assembly complex CIA proteins CIAO1 and MMS19. We show that serine-1940 is essential for the interaction between POLE1 and MMS19, but not POLE1 and CIAO1. No defect in either proliferation or survival was identified when POLE1 serine-1940 was mutated to alanine in human cells, even following treatment with DNA damaging agents. We conclude that serine-1940 phosphorylation and the interaction between serine-1940 and MMS19 are not essential functions in the C terminal domain of the catalytic subunit of DNA polymerase ε. PMID:27235625

Mature DIABLO/Smac Is Produced by the IMP Protease Complex on the Mitochondrial Inner Membrane

PubMed Central

Burri, Lena; Strahm, Yvan; Hawkins, Christine J.; Gentle, Ian E.; Puryer, Michelle A.; Verhagen, Anne; Callus, Bernard; Vaux, David; Lithgow, Trevor

2005-01-01

DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX5P in Imp1 and NX5S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space. PMID:15814844

Insights into cellulosome assembly and dynamics: from dissection to reconstruction of the supramolecular enzyme complex.

PubMed

Smith, Steven P; Bayer, Edward A

2013-10-01

Cellulosomes are multi-enzyme complexes produced by anaerobic bacteria for the efficient deconstruction of plant cell wall polysaccharides. The assembly of enzymatic subunits onto a central non-catalytic scaffoldin subunit is mediated by a highly specific interaction between the enzyme-bearing dockerin modules and the resident cohesin modules of the scaffoldin, which affords their catalytic activities to work synergistically. The scaffoldin also imparts substrate-binding and bacterial-anchoring properties, the latter of which involves a second cohesin-dockerin interaction. Recent structure-function studies reveal an ever-growing array of unique and increasingly complex cohesin-dockerin complexes and cellulosomal enzymes with novel activities. A 'build' approach involving multimodular cellulosomal segments has provided a structural model of an organized yet conformationally dynamic supramolecular assembly with the potential to form higher order structures. Copyright © 2013. Published by Elsevier Ltd.

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM.

PubMed

Zhou, Anna; Rohou, Alexis; Schep, Daniel G; Bason, John V; Montgomery, Martin G; Walker, John E; Grigorieff, Nikolaus; Rubinstein, John L

2015-10-06

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases.

Glycine Receptors Containing α2 or α3 Subunits Regulate Specific Ethanol-Mediated Behaviors

PubMed Central

Blednov, Yuri A.; Benavidez, Jillian M.; Black, Mendy; Leiter, Courtney R.; Osterndorff-Kahanek, Elizabeth

2015-01-01

Glycine receptors (GlyRs) are broadly expressed in the central nervous system. Ethanol enhances the function of brain GlyRs, and the GlyRα1 subunit is associated with some of the behavioral actions of ethanol, such as loss of righting reflex. The in vivo role of GlyRα2 and α3 subunits in alcohol responses has not been characterized despite high expression levels in the nucleus accumbens and amygdala, areas that are important for the rewarding properties of drugs of abuse. We used an extensive panel of behavioral tests to examine ethanol actions in mice lacking Glra2 (the gene encoding the glycine receptor alpha 2 subunit) or Glra3 (the gene encoding the glycine receptor alpha 3 subunit). Deletion of Glra2 or Glra3 alters specific ethanol-induced behaviors. Glra2 knockout mice demonstrate reduced ethanol intake and preference in the 24-hour two-bottle choice test and increased initial aversive responses to ethanol and lithium chloride. In contrast, Glra3 knockout mice show increased ethanol intake and preference in the 24-hour intermittent access test and increased development of conditioned taste aversion to ethanol. Mutants and wild-type mice consumed similar amounts of ethanol in the limited access drinking in the dark test. Other ethanol effects, such as anxiolysis, motor incoordination, loss of righting reflex, and acoustic startle response, were not altered in the mutants. The behavioral changes in mice lacking GlyRα2 or α3 subunits were distinct from effects previously observed in mice with knock-in mutations in the α1 subunit. We provide evidence that GlyRα2 and α3 subunits may regulate ethanol consumption and the aversive response to ethanol. PMID:25678534

Evidence for a remodelling of DNA-PK upon autophosphorylation from electron microscopy studies

PubMed Central

Morris, Edward P.; Rivera-Calzada, Angel; da Fonseca, Paula C. A.; Llorca, Oscar; Pearl, Laurence H.; Spagnolo, Laura

2011-01-01

The multi-subunit DNA-dependent protein kinase (DNA-PK), a crucial player in DNA repair by non-homologous end-joining in higher eukaryotes, consists of a catalytic subunit (DNA-PKcs) and the Ku heterodimer. Ku recruits DNA-PKcs to double-strand breaks, where DNA-PK assembles prior to DNA repair. The interaction of DNA-PK with DNA is regulated via autophosphorylation. Recent SAXS data addressed the conformational changes occurring in the purified catalytic subunit upon autophosphorylation. Here, we present the first structural analysis of the effects of autophosphorylation on the trimeric DNA-PK enzyme, performed by electron microscopy and single particle analysis. We observe a considerable degree of heterogeneity in the autophosphorylated material, which we resolved into subpopulations of intact complex, and separate DNA-PKcs and Ku, by using multivariate statistical analysis and multi-reference alignment on a partitioned particle image data set. The proportion of dimeric oligomers was reduced compared to non-phosphorylated complex, and those dimers remaining showed a substantial variation in mutual monomer orientation. Together, our data indicate a substantial remodelling of DNA-PK holo-enzyme upon autophosphorylation, which is crucial to the release of protein factors from a repaired DNA double-strand break. PMID:21450809

Activation of IKKalpha and IKKbeta through their fusion with HTLV-I tax protein.

PubMed

Xiao, G; Sun, S C

2000-10-26

Human T-cell leukemia virus type I (HTLV-I) Tax protein persistently stimulates the activity of IkappaB kinase (IKK), resulting in constitutive activation of the transcription factor NF-kappaB. Tax activation of IKK requires physical interaction of this viral protein with the IKK regulatory subunit, IKKgamma. The Tax/IKKgamma interaction allows Tax to engage the IKK catalytic subunits, IKKalpha and IKKbeta, although it remains unclear whether this linker function of IKKgamma is sufficient for supporting the Tax-specific IKK activation. To address this question, we have examined the sequences of IKKgamma required for modulating the Tax/IKK signaling. We demonstrate that when fused to Tax, a small N-terminal fragment of IKKgamma, containing its minimal IKKalpha/beta-binding domain, is sufficient for bringing Tax to and activating the IKK catalytic subunits. Disruption of the IKKalpha/beta-binding activity of this domain abolishes its function in modulating the Tax/IKK signaling. We further demonstrate that direct fusion of Tax to IKKalpha and IKKbeta leads to activation of these kinases. These findings suggest that the IKKgamma-directed Tax/IKK association serves as a molecular trigger for IKK activation.

Domain Architecture of the Catalytic Subunit in the ISW2-Nucleosome Complex▿

PubMed Central

Dang, Weiwei; Bartholomew, Blaine

2007-01-01

ATP-dependent chromatin remodeling has an important role in the regulation of cellular differentiation and development. For the first time, a topological view of one of these complexes has been revealed, by mapping the interactions of the catalytic subunit Isw2 with nucleosomal and extranucleosomal DNA in the complex with all four subunits of ISW2 bound to nucleosomes. Different domains of Isw2 were shown to interact with the nucleosome near the dyad axis, another near the entry site of the nucleosome, and another with extranucleosomal DNA. The conserved DEXD or ATPase domain was found to contact the superhelical location 2 (SHL2) of the nucleosome, providing a direct physical connection of ATP hydrolysis with this region of nucleosomes. The C terminus of Isw2, comprising the SLIDE (SANT-like domain) and HAND domains, was found to be associated with extranucleosomal DNA and the entry site of nucleosomes. It is thus proposed that the C-terminal domains of Isw2 are involved in anchoring the complex to nucleosomes through their interactions with linker DNA and that they facilitate the movement of DNA along the surface of nucleosomes. PMID:17908792

Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir

2010-10-08

Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-,more » and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.« less

AlphaII-spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts.

PubMed

Rotter, Björn; Bournier, Odile; Nicolas, Gael; Dhermy, Didier; Lecomte, Marie-Christine

2005-06-01

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of alpha- and beta-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent alpha-spectrin subunit in non-erythroid cells, alphaII-spectrin, contains two particular spectrin repeats in its central region, alpha9 and alpha10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the alpha9-alpha10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the alpha10 repeat of alphaII-spectrin whereas EVL interacts with the Src homology 3 domain located within the alpha9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between alphaII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

Escherichia coli F1Fo-ATP synthase with a b/δ fusion protein allows analysis of the function of the individual b subunits.

PubMed

Gajadeera, Chathurada S; Weber, Joachim

2013-09-13

The "stator stalk" of F1Fo-ATP synthase is essential for rotational catalysis as it connects the nonrotating portions of the enzyme. In Escherichia coli, the stator stalk consists of two (identical) b subunits and the δ subunit. In mycobacteria, one of the b subunits and the δ subunit are replaced by a b/δ fusion protein; the remaining b subunit is of the shorter b' type. In the present study, it is shown that it is possible to generate a functional E. coli ATP synthase containing a b/δ fusion protein. This construct allowed the analysis of the roles of the individual b subunits. The full-length b subunit (which in this case is covalently linked to δ in the fusion protein) is responsible for connecting the stalk to the catalytic F1 subcomplex. It is not required for interaction with the membrane-embedded Fo subcomplex, as its transmembrane helix can be removed. Attachment to Fo is the function of the other b subunit which in turn has only a minor (if any at all) role in binding to δ. Also in E. coli the second b subunit can be shortened to a b' type.

Dietary phenolic compounds selectively inhibit the individual subunits of maltase-glucoamylase and sucrase-isomaltase with the potential of modulating glucose release

USDA-ARS?s Scientific Manuscript database

In this study, it was hypothesized that dietary phenolic compounds selectively inhibit the individual C- and N-terminal (Ct, Nt) subunits of the two small intestinal alpha-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), for a modulated glycemic carbohydrate digestion. The inhi...

How does cellulosome composition influence deconstruction of lignocellulosic substrates in Clostridium (Ruminiclostridium) thermocellum DSM 1313?

PubMed

Yoav, Shahar; Barak, Yoav; Shamshoum, Melina; Borovok, Ilya; Lamed, Raphael; Dassa, Bareket; Hadar, Yitzhak; Morag, Ely; Bayer, Edward A

2017-01-01

Bioethanol production processes involve enzymatic hydrolysis of pretreated lignocellulosic biomass into fermentable sugars. Due to the relatively high cost of enzyme production, the development of potent and cost-effective cellulolytic cocktails is critical for increasing the cost-effectiveness of bioethanol production. In this context, the multi-protein cellulolytic complex of Clostridium ( Ruminiclostridium ) thermocellum, the cellulosome, was studied here. C. thermocellum is known to assemble cellulosomes of various subunit (enzyme) compositions, in response to the available carbon source. In the current study, different carbon sources were used, and their influence on both cellulosomal composition and the resultant activity was investigated. Glucose, cellobiose, microcrystalline cellulose, alkaline-pretreated switchgrass, alkaline-pretreated corn stover, and dilute acid-pretreated corn stover were used as sole carbon sources in the growth media of C. thermocellum strain DSM 1313. The purified cellulosomes were compared for their activity on selected cellulosic substrates. Interestingly, cellulosomes derived from cells grown on lignocellulosic biomass showed no advantage in hydrolyzing the original carbon source used for their production. Instead, microcrystalline cellulose- and glucose-derived cellulosomes were equal or superior in their capacity to deconstruct lignocellulosic biomass. Mass spectrometry analysis revealed differential composition of catalytic and structural subunits (scaffoldins) in the different cellulosome samples. The most abundant catalytic subunits in all cellulosome types include Cel48S, Cel9K, Cel9Q, Cel9R, and Cel5G. Microcrystalline cellulose- and glucose-derived cellulosome samples showed higher endoglucanase-to-exoglucanase ratios and higher catalytic subunit-per-scaffoldin ratios compared to lignocellulose-derived cellulosome types. The results reported here highlight the finding that cellulosomes derived from cells grown on glucose and microcrystalline cellulose are more efficient in their action on cellulosic substrates than other cellulosome preparations. These results should be considered in the future development of C. thermocellum -based cellulolytic cocktails, designer cellulosomes, or engineering of improved strains for deconstruction of lignocellulosic biomass.

5' adenosine monophosphate-activated protein kinase, metabolism and exercise.

PubMed

Aschenbach, William G; Sakamoto, Kei; Goodyear, Laurie J

2004-01-01

The 5' adenosine monophosphate-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that functions as a metabolic 'fuel gauge' in skeletal muscle. AMPK is a ubiquitous heterotrimeric protein, consisting of an alpha catalytic, and beta and gamma regulatory subunits that exist in multiple isoforms and are all required for full enzymatic activity. During exercise, AMPK becomes activated in skeletal muscle in response to changes in cellular energy status (e.g. increased adenosine monophosphate [AMP]/adenosine triphosphate [ATP] and creatine/phosphocreatine ratios) in an intensity-dependent manner, and serves to inhibit ATP-consuming pathways, and activate pathways involved in carbohydrate and fatty-acid metabolism to restore ATP levels. Recent evidence shows that although AMPK plays this key metabolic role during acute bouts of exercise, it is also an important component of the adaptive response of skeletal muscles to endurance exercise training because of its ability to alter muscle fuel reserves and expression of several exercise-responsive genes. This review discusses the putative roles of AMPK in acute and chronic exercise responses, and suggests avenues for future AMPK research in exercise physiology and biochemistry.

Congenitally learned helpless rats show abnormalities in intracellular signaling.

PubMed

Kohen, Ruth; Neumaier, John F; Hamblin, Mark W; Edwards, Emmeline

2003-03-15

Affective disorders and the drugs used to treat them lead to changes in intracellular signaling. We used a genetic animal model to investigate to what extent changes in intracellular signal transduction confer a vulnerability to mood or anxiety disorders. Levels of gene expression in a selectively bred strain of rats with a high vulnerability to develop congenitally learned helplessness (cLH), a strain highly resistant to the same behavior (cNLH) and outbred Sprague-Dawley (SD) control animals were compared using quantitative reverse transcription polymerase chain reaction. Congenitally learned helpless animals had a 24%-30% reduced expression of the cyclic adenosine monophosphate response element binding protein messenger ribonucleic acid (mRNA) in the hippocampus and a 40%-41% increased level of the antiapoptotic protein bcl-2 mRNA in the prefrontal cortex compared to cNLH and SD rats. Other significant changes included changes in the expression levels of the alpha catalytic subunit of protein kinase A, glycogen synthase kinase 3beta, and protein kinase C epsilon. Congenitally learned helpless animals show evidence of altered signal transduction and regulation of apoptosis compared to cNLH and SD control animals.

Hybrid fusions show that inter-monomer electron transfer robustly supports cytochrome bc{sub 1} function in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ekiert, Robert; Czapla, Monika; Sarewicz, Marcin

2014-08-22

Highlights: • We used hybrid fusion bc{sub 1} complex to test inter-monomer electron transfer in vivo. • Cross-inactivated complexes were able to sustain photoheterotrophic growth. • Inter-monomer electron transfer supports catalytic cycle in vivo. • bc{sub 1} dimer is functional even when cytochrome b subunits come from different species. - Abstract: Electronic connection between Q{sub o} and Q{sub i} quinone catalytic sites of dimeric cytochrome bc{sub 1} is a central feature of the energy-conserving Q cycle. While both the intra- and inter-monomer electron transfers were shown to connect the sites in the enzyme, mechanistic and physiological significance of the lattermore » remains unclear. Here, using a series of mutated hybrid cytochrome bc{sub 1}-like complexes, we show that inter-monomer electron transfer robustly sustains the function of the enzyme in vivo, even when the two subunits in a dimer come from different species. This indicates that minimal requirement for bioenergetic efficiency is to provide a chain of cofactors for uncompromised electron flux between the catalytic sites, while the details of protein scaffold are secondary.« less

Rubisco small subunits from the unicellular green alga Chlamydomonas complement Rubisco-deficient mutants of Arabidopsis.

PubMed

Atkinson, Nicky; Leitão, Nuno; Orr, Douglas J; Meyer, Moritz T; Carmo-Silva, Elizabete; Griffiths, Howard; Smith, Alison M; McCormick, Alistair J

2017-04-01

Introducing components of algal carbon concentrating mechanisms (CCMs) into higher plant chloroplasts could increase photosynthetic productivity. A key component is the Rubisco-containing pyrenoid that is needed to minimise CO 2 retro-diffusion for CCM operating efficiency. Rubisco in Arabidopsis was re-engineered to incorporate sequence elements that are thought to be essential for recruitment of Rubisco to the pyrenoid, namely the algal Rubisco small subunit (SSU, encoded by rbcS) or only the surface-exposed algal SSU α-helices. Leaves of Arabidopsis rbcs mutants expressing 'pyrenoid-competent' chimeric Arabidopsis SSUs containing the SSU α-helices from Chlamydomonas reinhardtii can form hybrid Rubisco complexes with catalytic properties similar to those of native Rubisco, suggesting that the α-helices are catalytically neutral. The growth and photosynthetic performance of complemented Arabidopsis rbcs mutants producing near wild-type levels of the hybrid Rubisco were similar to those of wild-type controls. Arabidopsis rbcs mutants expressing a Chlamydomonas SSU differed from wild-type plants with respect to Rubisco catalysis, photosynthesis and growth. This confirms a role for the SSU in influencing Rubisco catalytic properties. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation.

PubMed

Støve, Svein Isungset; Magin, Robert S; Foyn, Håvard; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas

2016-07-06

N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae*

PubMed Central

Nedielkov, Ruslan; Steffen, Wojtek; Steuber, Julia; Möller, Heiko M.

2013-01-01

The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na+-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na+-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na+-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na+-NQR. PMID:24003222

The Thiamin Pyrophosphate-Motif

NASA Technical Reports Server (NTRS)

Dominiak, P.; Ciszak, E.

2003-01-01

Using databases the authors have identified a common thiamin pyrophosphate (TPP)-motif in the family of functionally diverse TPP-dependent enzymes. This common motif consists of multimeric organization of subunits and two catalytic centers. Each catalytic center (PP:PYR) is formed at the interface of the PP-domain binding the magnesium ion, pyrophosphate and amhopyrimidine ring of TPP, and the PYR-domain binding the aminopyrimidine ring of that cofactor. A pair of these catalytic centers constitutes the catalytic core (PP:PYR)(sub 2) within these enzymes. Analysis of the structural elements of this catalytic core reveals novel definition of the common amino acid sequences, which are GXPhiX(sub 4)(G)PhiXXGQ and GDGX(sub 25-30)NN in the PP-domain, and the EX(sub 4)(G)PhiXXGPhi in the PYR-domain, where Phi corresponds to a hydrophobic amino acid. This TPP-motif provides a novel tool for annotation of TPP-dependent enzymes useful in advancing functional proteomics.

Cloning, Purification, and Characterization of a Heterodimeric β-Galactosidase from Lactobacillus kefiranofaciens ZW3.

PubMed

He, Xi; Han, Ning; Wang, Yan-Ping

2016-01-01

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60°C and 7.0, and 50°C and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

Differential processing of the two subunits of human choriogonadotropin (hCG) by granulosa cells. I. Preparation and characterization of selectively labeled hCG

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landefeld, T.D.; Byrne, M.D.; Campbell, K.L.

1981-12-01

The alpha- and beta-subunits of hCG were radioiodinated and recombined with unlabeled complementary subunits. The resultant recombined hormones, selectively labeled in either the alpha- or beta-subunit, were separated from unrecombined subunit by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, extracted with Triton X-100, and characterized by binding analysis. The estimates of maximum binding (active fraction) of the two resultant selectively labeled, recombined hCG preparations, determined with excess receptor were 0.41 and 0.59. These values are similar to those obtained when hCG is labeled as an intact molecule. The specific activities of the recombined preparations were estimated by four different methods, and themore » resulting values were used in combination with the active fraction estimates to determine the concentrations of active free and bound hormone. Binding analyses were run using varying concentrations of both labeled and unlabeled hormone. Estimates of the equilibrium dissociation binding constant (Kd) and receptor capacity were calculated in three different ways. The mean estimates of capacity (52.6 and 52.7 fmol/mg tissue) and Kd (66.6 and 65.7 pM) for the two preparations were indistinguishable. Additionally, these values were similar to values reported previously for hCG radioiodinated as an intact molecule. The availability of well characterized, selectively labeled hCG preparations provides new tools for studying the mechanism of action and the target cell processing of the subunits of this hormone.« less

Widespread Distribution and Functional Specificity of the Copper Importer CcoA: Distinct Cu Uptake Routes for Bacterial Cytochrome c Oxidases

DOE PAGES

Khalfaoui-Hassani, Bahia; Wu, Hongjiang; Blaby-Haas, Crysten E.; ...

2018-02-27

ABSTRACT Cytochromecoxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (Cu B) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the Cu Batom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacteriumRhodobacter capsulatusindicated that the copper atom needed for the Cu Bsite ofcbb 3-type cytochromecoxidase (cbb 3-Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis ofmore » CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochromecoxidases. To define the specificity of CcoA activity, we investigated its function inRhodobacter sphaeroides, a close relative ofR. capsulatusthat contains bothcbb 3- andaa 3-Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production ofcbb 3-Cox and not that ofaa 3-Cox was affected. We therefore concluded that CcoA is dedicated solely tocbb 3-Cox biogenesis, establishing that distinct copper uptake systems provide the Cu Batoms to the catalytic sites of these two similar cytochromecoxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions. IMPORTANCEThecbb 3- andaa 3-type cytochromecoxidases belong to the widespread heme-copper oxidase superfamily. They are membrane-integral cuproproteins that catalyze oxygen reduction to water under hypoxic and normoxic growth conditions. These enzymes diverge in terms of subunit and cofactor composition, yet they all share a conserved heme-copper binuclear site within their catalytic subunit. In this study, we show that the copper atoms of the catalytic center of two similar cytochromecoxidases from this superfamily are provided by different copper uptake systems during their biogenesis. This finding illustrates different strategies by which organisms fine-tune the trafficking of copper, which is an essential but toxic micronutrient.« less

Widespread Distribution and Functional Specificity of the Copper Importer CcoA: Distinct Cu Uptake Routes for Bacterial Cytochrome c Oxidases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khalfaoui-Hassani, Bahia; Wu, Hongjiang; Blaby-Haas, Crysten E.

ABSTRACT Cytochromecoxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (Cu B) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the Cu Batom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacteriumRhodobacter capsulatusindicated that the copper atom needed for the Cu Bsite ofcbb 3-type cytochromecoxidase (cbb 3-Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis ofmore » CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochromecoxidases. To define the specificity of CcoA activity, we investigated its function inRhodobacter sphaeroides, a close relative ofR. capsulatusthat contains bothcbb 3- andaa 3-Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production ofcbb 3-Cox and not that ofaa 3-Cox was affected. We therefore concluded that CcoA is dedicated solely tocbb 3-Cox biogenesis, establishing that distinct copper uptake systems provide the Cu Batoms to the catalytic sites of these two similar cytochromecoxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions. IMPORTANCEThecbb 3- andaa 3-type cytochromecoxidases belong to the widespread heme-copper oxidase superfamily. They are membrane-integral cuproproteins that catalyze oxygen reduction to water under hypoxic and normoxic growth conditions. These enzymes diverge in terms of subunit and cofactor composition, yet they all share a conserved heme-copper binuclear site within their catalytic subunit. In this study, we show that the copper atoms of the catalytic center of two similar cytochromecoxidases from this superfamily are provided by different copper uptake systems during their biogenesis. This finding illustrates different strategies by which organisms fine-tune the trafficking of copper, which is an essential but toxic micronutrient.« less

The Glucoamylase Inhibitor Acarbose Is a Direct Activator of Phosphorylase Kinase

PubMed Central

Nadeau, Owen W.; Liu, Weiya; Boulatnikov, Igor G.; Sage, Jessica M.; Peters, Jennifer L.; Carlson, Gerald M.

2011-01-01

Phosphorylase kinase (PhK), an (αβγδ)4 complex, stimulates energy production from glycogen in the cascade activation of glycogenolysis. Its large homologous α and β subunits regulate the activity of the catalytic γ subunit and account for 81% of PhK’s mass. Both subunits are thought to be multi-domain structures, and recent predictions based on their sequences suggest the presence of potentially functional glucoamylase (GH15)-like domains near their amino-termini. We present the first experimental evidence for such a domain in PhK, by demonstrating that the glucoamylase inhibitor acarbose binds PhK, perturbs its structure, and stimulates its kinase activity. PMID:20604537

The role of nicotinic receptor alpha 7 subunits in nicotine discrimination.

PubMed

Stolerman, I P; Chamberlain, S; Bizarro, L; Fernandes, C; Schalkwyk, L

2004-03-01

The subtypes of nicotinic receptors at which the behavioural effects of nicotine originate are not fully understood. The experiments described here use mice lacking the alpha7 subunit of nicotinic receptors to investigate the role of alpha7-containing receptors in nicotine discrimination. Wild-type and alpha7-knockout mice were trained in a two-lever nicotine discrimination procedure using a tandem schedule of food reinforcement. Mutant mice exhibited baseline rates of lever-pressing as low as 52.2% of rates in wild-type controls (n=21-24). Mutant and wild-type mice acquired discrimination of nicotine (0.4 or 0.8 mg/kg) at a similar rate (n=10-12) and reached similar final levels of accuracy (71.9 +/- 4.4% and 90.8 +/- 3.1% after 60 training sessions for 0.4 and 0.8 mg/kg training doses, respectively, in mutant mice, as compared with 75.0 +/- 6.5% and 87.6 +/- 4.8% for wild types). The genotypes exhibited similar steep dose-response curves for nicotine discrimination. In both genotypes, dose-response curves for mice trained with 0.8 mg/kg of nicotine were displaced three- to four-fold to the right as compared with those for the mice trained with the smaller dose. The predominant effect of nicotine on the overall rate of responding was a reduction at the largest doses tested and there was no difference between the genotypes. The results suggest that nicotinic receptors containing the alpha7 subunit do not contribute to the discriminative stimulus or response-rate-depressant effects of nicotine, although they may regulate baseline rates of operant responding.

The gene for the alpha 1 subunit of the skeletal muscle dihydropyridine-sensitive calcium channel (Cchl1a3) maps to mouse chromosome 1.

PubMed

Chin, H; Krall, M; Kim, H L; Kozak, C A; Mock, B

1992-12-01

Cchl1a3 encodes the dihydropyridine-sensitive calcium channel alpha 1 subunit isoform predominantly expressed in skeletal muscle. mdg (muscular dysgenesis) has previously been implicated as a mutant allele of this gene. Hybridization of a rat brain cDNA probe for Cchl1a3 to Southern blots of DNAs from a panel of Chinese hamster x mouse somatic cell hybrids suggested that this gene maps to mouse Chromosome 1. Analysis of the progeny of an inbred strain cross-positioned Cchl1a3 1.3 cM proximal to the Pep-3 locus on Chr 1.

The effects of brefeldin-A on the high mannose oligosaccharides of mouse thyrotropin, free alpha-subunits, and total glycoproteins.

PubMed

Perkel, V S; Liu, A Y; Miura, Y; Magner, J A

1988-07-01

We have studied the effects of Brefeldin-A (BFA) on the processing of high mannose (Man) oligosaccharides of TSH. BFA is a drug that inhibits the intracellular translocation of newly synthesized glycoproteins and causes dilatation of the rough endoplasmic reticulum (RER) as well as mild swelling of the Golgi apparatus. Mouse pituitary thyrotropic tumor tissue was incubated with [3H]Man for a 2-h pulse, with and without a 3-h chase; BFA (5 micrograms/ml) was included during selected pulse and selected chase incubations. TSH and free alpha-subunits were obtained from detergent lysates of tissue by immunoprecipitation using specific antisera. Total glycoproteins were obtained by trichloroacetic acid precipitation. Endoglycosidase-H-released [3H]oligosaccharides were analyzed by paper chromatography. BFA inhibited carbohydrate processing of TSH, free alpha-subunits, and total glycoproteins, resulting in the accumulation of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2, especially during the chase period. Subcellular fractions enriched in RER, heavy (proximal) Golgi, and light (distal) Golgi were prepared by centrifugation in discontinuous sucrose gradients. [3H]Man-labeled oligosaccharides of TSH and total glycoproteins in the subcellular fractions were analyzed. In contrast to oligosaccharides with eight or nine Man residues found in control incubations, BFA caused the accumulation of oligosaccharides containing five to eight Man residues. These BFA-induced oligosaccharide alterations began in the RER and proximal Golgi with the 2-h pulse and extended into the distal Golgi during the chase incubations. Thus, BFA blocks the normal intracellular transport and processing of TSH, free alpha-subunits, and total glycoproteins within thyrotrophs, causing species with smaller than normal high Man oligosaccharides to appear in subcellular compartments as early as the RER. The translocation block between RER and Golgi produced by BFA may prevent the processing of Man8GlcNAc2 to Man5GlcNAc2 by Golgi (alpha,1-2)mannosidase I, yet the species retained within the RER may be subject to ongoing processing by endoplasmic reticulum (alpha,1-2)mannosidase, resulting in the accumulation of Man5-8GlcNAc2 within the RER.

Eukaryotic polypeptide elongation system and its sensitivity to the inhibitory substances of plant origin.

PubMed

Gałasiński, W

1996-05-01

The structural and functional characteristics of the elongation system (ribosomes and elongation factors) are presented. The immunochemical and diagnostic meaning of the ribosome investigations is considered. Evidence of the participation of ribosomes in the first step of protein glycosylation is presented. The heterogeneous elongation factor eEF-1, isolated from Guerin epithelioma, can be separated into three fractions: one of them functionally corresponds to EF-1 alpha, the second on to EF-1 beta gamma, and the third is an unidentified, active aggregate named EF-1B, which contains the subunit forms EF-1 alpha and EF-1 beta gamma, and other polypeptides showing protein kinase activity. The aggregate EF-1B can be autophosphorylated, while the subunit forms EF-1 alpha and EF-1 beta gamma can neither become autophosphorylated nor phosphorylate other polypeptides. The subunit form EF-beta gamma consists from two polypeptides of 32 and 51 kDa, corresponding to other eukaryotic beta and gamma polypeptides, respectively. EF-1 beta gamma is thermostable and protects against thermal inactivation of EF-1 alpha in the EF-1 alpha-EF-1 beta gamma complex. Pure eEF-2 preparations isolated from normal and neoplastic tissues show different structural features. The existence of eEF-2 in multiple forms, differing in molecular mass, have been found. The eEF-2 with molecular weight of about 100 kDa can be phosphorylated, while eEF-2 of about 65 kDa was not phosphorylated by protein kinase eEF-2. The phosphorylated eEF-2 lost its activity, and this effect was reversed by dephosphorylation. The eEF-2 (65 kDa) was isolated from the active polyribosomes, and it may directly participate in the translocation step of the peptide elongation. It was noted that the components of elongation system can be inhibited, in separate steps, by the substances isolated from various sources of plant origin. Alkaloids emetine and cepheline, cardiac remedy digoxin, saponin glycoside, and its aglycon directly inactivated ribosomes. Quercetin inhibited eEF-1 activity by directly influencing its subunit form EF-1 alpha. eEF-2 was shown to be a target site of the inhibitory action of the glycoside isolated from Melissa officinalis leaves.

IGF-1-dependent subunit communication of the IGF-1 holoreceptor: Interactions between. alpha. beta. heterodimeric receptor halves

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilden, P.A.; Treadway, J.L.; Morrison, B.D.

1989-12-12

Examination of {sup 125}I-IGF-1 affinity cross-linking and {beta}-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated {alpha}{beta} heterodimeric IGF-1 receptors into an {alpha}{sub 2}{beta}{sub 2} heterotetrameric state, in a similar manner to that observed for the insulin receptor. The formation of the {alpha}{sub 2}{beta}{sub 2} heterotetrameric IGF-1 receptor complex from the partially purified {alpha}{beta} heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified {alpha}{beta} heterodimers into an {alpha}{sub 2}{beta}{sub 2} heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulatemore » the protein kinase activity of the purified {alpha}{beta} heterodimeric insulin receptor complex. Incubation of the {alpha}{sub 2}{beta}{sub 2} heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter {sup 125}I-IGF-1 binding or IGF-1 stimulation of protein kinase activity. However, IAN treatment of the {alpha}{beta} heterodimeric IGF-1 receptors inhibited the IGF-1 dependent covalent formation of the disulfide-linked {alpha}{sub 2}{beta}{sub 2} heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated {alpha}{beta} heterodimeric IGF-1 receptor complexes into a disulfide-linked {alpha}{sub 2}{beta}{sub 2} heterotetrameric state whereas Mn/MgATP induces a noncovalent association. Therefore, unlike the insulin receptor in which noncovalent association is sufficient for kinase activation, only the covalent assembly of the IGF-1 receptor {alpha}{beta} heterodimers into the {alpha}{sub 2}{beta}{sub 2} heterotetrameric holoreceptor complex is associated with ligand-stimulated protein kinase activation.« less

Absence of integrin alpha 7 causes a novel form of muscular dystrophy.

PubMed

Mayer, U; Saher, G; Fässler, R; Bornemann, A; Echtermeyer, F; von der Mark, H; Miosge, N; Pöschl, E; von der Mark, K

1997-11-01

Integrin alpha 7 beta 1 is a specific cellular receptor for the basement membrane protein laminin-1 (refs 1,2), as well as for the laminin isoforms -2 and -4 (ref. 3). The alpha 7 subunit is expressed mainly in skeletal and cardiac muscle and has been suggested to be involved in differentiation and migration processes during myogenesis. Three cytoplasmic and two extracellular splice variants that have been described are developmentally regulated and expressed in different sites in the muscle. In adult muscle, the alpha 7A and alpha 7B subunits are concentrated in myotendinous junctions but can also be detected in neuromuscular junctions and along the sarcolemmal membrane. To study the potential involvement of alpha 7 integrin, during myogenesis and its role in muscle integrity and function, we generated a null allele of the alpha 7 gene (Itga7) in the germline of mice by homologous recombination in embryonic stem (ES) cells. Surprisingly, mice homozygous for the mutation are viable and fertile, indicating that the alpha 7 beta 1 integrin is not essential for myogenesis. However, histological analysis of skeletal muscle revealed typical symptoms of a progressive muscular dystrophy starting soon after birth, but with a distinct variability in different muscle types. The observed histopathological changes strongly indicate an impairment of function of the myotendinous junctions. These findings demonstrate that alpha 7 beta 1 integrin represents an indispensable linkage between the muscle fibre and the extracellular matrix that is independent of the dystrophin-dystroglycan complex-mediated interaction of the cytoskeleton with the muscle basement membrane.

Molecular cloning and characterization of G alpha proteins from the western tarnished plant bug, Lygus hesperus

USDA-ARS?s Scientific Manuscript database

The G-alpha subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-base...

Interferon Antagonism as a Common Virulence Factor of Hemorrhagic Fever Viruses

DTIC Science & Technology

2008-02-01

S. Prehn , A. Leutz, H. Haller, and E. Hartmann. 1997. Cloning of two novel human importin-alpha subunits and analysis of the expression pattern of...the importin-alpha protein family. FEBS Lett 417:104-8. 12. Kohler, M., C. Speck, M. Christiansen, F. R. Bischoff, S. Prehn , H. Haller, D. Gorlich

Identification of a novel bursicon-regulated transcriptional regulator, md133790, in house fly Musca domestica

USDA-ARS?s Scientific Manuscript database

Bursicon is a neuropeptide that regulates cuticle sclerotization (hardening and tanning) and wing expansion in insects via a G-protein coupled receptor. The peptide consists of alpha and beta subunits. In the present study, we cloned bursicon alpha and beta genes in the house fly Musca domestica us...

Definition of the mutation responsible for maple syrup urine disease in Poll Shorthorns and genotyping Poll Shorthorns and Poll Herefords for maple syrup urine disease alleles.

PubMed

Dennis, J A; Healy, P J

1999-08-01

The organisation of the E1alpha subunit of bovine branched-chain alpha-keto acid dehydrogenase gene was established. c DNA was cloned from Poll Shorthorn x Poll Hereford calves affected with Maple Syrup Urine Disease to identify the mutation responsible for the disease in Poll Shorthorns. Clones containing the c DNA sequences inherited from the Poll Shorthorn sire of the affected calves were identified. Paternal clones were sequenced and a cytidine to thymidine transition was found at nucleotide 1380. The mutation is predicted to substitute leucine in place of a highly conserved proline at codon 372. A polymerase chain reaction procedure was developed for detection of the 1380C-->T mutation in genomic DNA. Three Poll Shorthorn parents of affected calves and three affected Poll Shorthorn x Poll Hereford calves were heterozygous and an affected Poll Shorthorn calf was homozygous for this mutation. An improved polymerase chain reaction procedure was also devised to genotype Poll Herefords for the 248C-->T mutation. The procedures will facilitate disease prevention programs and assist in differential diagnosis of conditions in new-born calves that present with a rapid onset of progressive neurological disease and are characterised histologically by 'status spongiosus'. Maple Syrup Urine Disease (MSUD) is an autosomal recessive defect reported in humans (Danner and Elsas 1989), and in Poll Hereford (PH) and Poll Shorthorn (PS) calves (Harper et al 1986, Healy et al 1992). The clinical, biochemical and pathological manifestations of the disease are identical in the two breeds of cattle, and are characterised by the rapid onset of progressive neurological disease, leading to death within a few days of birth. The disease is caused by a deficiency of activity of the mitochondrial enzyme branched-chain alpha-keto acid dehydrogenase (BCKADH). This deficiency leads to elevated concentrations, in blood and tissues, of branched chain alpha-keto acids and their precursors, the branched chain amino acids, valine, leucine and isoleucine. BCKADH consists of four subunits E1alpha, E1beta, E2 and E3 that are encoded by separate genes, and MSUD may result from deficiency of any of the subunits. In PH s, the disease in caused by premature termination of translation, of the E1alpha subunit, that is induced by a cytidine to thymidine transition exon 2 (248C-->T), that converts the glutamine codon -6 to a stop codon (Q-6ST; Zhang et al 1990). We have shown that MSUD -affected PSxPH calves are heterozygous at the PH locus, illustrating molecular heterogeneity exists for bovine MSUD (Healy and Dennis 1994a). The fact that these crossbred calves are affected, indicates the PS, like the PH mutation, resides in the E1alpha subunit. Copyright 1999 Harcourt Publishers Ltd.

Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR Is Essential for PROTEIN PHOSPHATASE 2A Holoenzyme Assembly and Plays Important Roles in Hormone Signaling, Salt Stress Response, and Plant Development1[W][OPEN

PubMed Central

Chen, Jian; Hu, Rongbin; Zhu, Yinfeng; Shen, Guoxin; Zhang, Hong

2014-01-01

PROTEIN PHOSPHATASE 2A (PP2A) is a major group of serine/threonine protein phosphatases in eukaryotes. It is composed of three subunits: scaffolding subunit A, regulatory subunit B, and catalytic subunit C. Assembly of the PP2A holoenzyme in Arabidopsis (Arabidopsis thaliana) depends on Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR (AtPTPA). Reduced expression of AtPTPA leads to severe defects in plant development, altered responses to abscisic acid, ethylene, and sodium chloride, and decreased PP2A activity. In particular, AtPTPA deficiency leads to decreased methylation in PP2A-C subunits (PP2Ac). Complete loss of PP2Ac methylation in the suppressor of brassinosteroid insensitive1 mutant leads to 30% reduction of PP2A activity, suggesting that PP2A with a methylated C subunit is more active than PP2A with an unmethylated C subunit. Like AtPTPA, PP2A-A subunits are also required for PP2Ac methylation. The interaction between AtPTPA and PP2Ac is A subunit dependent. In addition, AtPTPA deficiency leads to reduced interactions of B subunits with C subunits, resulting in reduced functional PP2A holoenzyme formation. Thus, AtPTPA is a critical factor for committing the subunit A/subunit C dimer toward PP2A heterotrimer formation. PMID:25281708

Evidence that assembly of the yeast cytochrome bc1 complex involves formation of a large core structure in the inner mitochondrial membrane

PubMed Central

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L.

2009-01-01

The assembly status of the cytochrome bc1 complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc1 subunits had been deleted. In all the yeast strains tested a bc1 sub-complex of about 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS-PAGE and immunodecoration, revealed the presence of the two catalytic subunits cytochrome b and cytochrome c1, associated with the non catalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Altogether these bc1 subunits build up the core structure of the cytochrome bc1 complex which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc1 core structure may represent a true assembly intermediate during the maturation of the bc1 complex, first because of its wide distribution in distinct yeast deletion strains and second for its characteristics of stability which resemble those of the intact homodimeric bc1 complex. Differently from this latter, however, the bc1 core structure is not able to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc1 complex provides a number of new elements for clarification of the molecular events leading to the maturation of the yeast cytochrome bc1 complex in the inner mitochondrial membrane. PMID:19236481

DIRECT MODULATION OF THE PROTEIN KINASE A CATALYTIC SUBUNIT α BY GROWTH FACTOR RECEPTOR TYROSINE KINASES

PubMed Central

Caldwell, George B.; Howe, Alan K.; Nickl, Christian K.; Dostmann, Wolfgang R.; Ballif, Bryan A.; Deming, Paula B.

2011-01-01

The cyclic-AMP-dependent protein kinase A (PKA) regulates processes such as cell proliferation and migration following activation of growth factor receptor tyrosine kinases (RTKs), yet the signaling mechanisms that link PKA with growth factor receptors remain largely undefined. Here we report that RTKs can directly modulate the function of the catalytic subunit of PKA (PKA-C) through post-translational modification. In vitro kinase assays revealed that both the epidermal growth factor and platelet derived growth factor receptors (EGFR and PDGFR, respectively) tyrosine phosphorylate PKA-C. Mass spectrometry identified tyrosine 330 (Y330) as a receptor-mediated phosphorylation site and mutation of Y330 to phenylalanine (Y330F) all but abolished the RTK-mediated phosphorylation of PKA-C in vitro. Y330 resides within a conserved region at the C-terminal tail of PKA-C that allosterically regulates enzymatic activity. Therefore, the effect of phosphorylation at Y330 on the activity of PKA-C was investigated. The Km for a peptide substrate was markedly decreased when PKA-C subunits were tyrosine phosphorylated by the receptors as compared to un-phosphorylated controls. Importantly, tyrosine-phosphorylated PKA-C subunits were detected in cells stimulated with EGF, PDGF and FGF2 and in fibroblasts undergoing PDGF-mediated chemotaxis. These results demonstrate a direct, functional interaction between RTKs and PKA-C and identify tyrosine phosphorylation as a novel mechansim for regulating PKA activity. PMID:21866565

Overexpression of stress-inducible OsBURP16, the β subunit of polygalacturonase 1, decreases pectin content and cell adhesion and increases abiotic stress sensitivity in rice.

PubMed

Liu, Huanhuan; Ma, Yan; Chen, Na; Guo, Siyi; Liu, Huili; Guo, Xiaoyu; Chong, Kang; Xu, Yunyuan

2014-05-01

Polygalacturonase (PG), one of the hydrolases responsible for cell wall pectin degradation, is involved in organ consenescence and biotic stress in plants. PG1 is composed of a catalytic subunit, PG2, and a non-catalytic PG1β subunit. OsBURP16 belongs to the PG1β-like subfamily of BURP-family genes and encodes one putative PG1β subunit precursor in rice (Oryza sativa L.). Transcription of OsBURP16 is induced by cold, salinity and drought stresses, as well as by abscisic acid (ABA) treatment. Analysis of plant survival rates, relative ion leakage rates, accumulation levels of H2 O2 and water loss rates of leaves showed that overexpression of OsBURP16 enhanced sensitivity to cold, salinity and drought stresses compared with controls. Young leaves of Ubi::OsBURP16 transgenic plants showed reduced cell adhesion and increased cuticular transpiration rate. Mechanical strength measurement of Ubi::OsBURP16 plants showed that reduced force was required to break leaves as compared with wild type. Transgenic rice showed enhanced PG activity and reduced pectin content. All these results suggested that overexpression of OsBURP16 caused pectin degradation and affected cell wall integrity as well as transpiration rate, which decreased tolerance to abiotic stresses. © 2013 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.

A functional thyrotropin- and growth hormone-secreting pituitary adenoma with a ultrastructurally monomorphic feature: a case study.

PubMed

Ozawa, Y; Kameya, T; Kasuga, A; Naritaka, H; Kanda, N; Maruyama, H; Saruta, T

1998-04-01

A 38-yr-old female with a TSH- and GH-secreting pituitary adenoma is described, who had both overt symptoms, hyperthyroidism and acromegaly. Her serum TSH was not suppressed despite high concentrations of free T3 and free T4, and her alpha-subunit/TSH molar ratio was high. Her serum GH was consistently high, and was not suppressed by an oral glucose tolerance test. Preoperative testing revealed that, although the TSH response was impaired, TSH, alpha-subunit and GH were increased by TRH injection, and that these hormones were reduced by bromocriptine or somatostatin analog. Although she did not have hyperprolactinemia, the in vitro culture and immunohistochemical studies revealed that the adenoma cells produced and released PRL, in addition to TSH, alpha-subunit and GH. Immunohistochemical studies showed the presence of GH in the cytoplasm of many adenoma cells. TSH beta-positive adenoma cells were less frequently seen than GH-positive adenoma cells. No cells showed the coexistence of GH and TSH beta, and a few cells were positive for PRL. By electron microscopy, the adenoma was found to be composed of a single cell type resembling thyrotrophs, and did not have any characteristics of somatotrophs. This case was considered to be of interest, because the adenoma was ultrastructurally monomorphous, but immunohistochemically polymorphous.

Contribution of oligosaccharides to protection of the H,K-ATPase beta-subunit against trypsinolysis.

PubMed

Crothers, James M; Asano, Shinji; Kimura, Tohru; Yoshida, Ayumi; Wong, Aline; Kang, Jung Wook; Forte, John G

2004-08-01

The proton-pumping H+,K+-adenosinetriphosphatase (H,K-ATPase), responsible for acid secretion by the gastric parietal cell, faces a harshly acidic environment, with some pepsin from neighboring chief cells, at its luminal surface. Its large catalytic alpha-subunit is mostly oriented cytoplasmically. The smaller beta-subunit (HKbeta), is mainly extracellular, with one transmembrane domain and a small cytoplasmic domain. Seven N-linked oligosaccharides in the extracellular domain of HKbeta are thought to contribute to protection of the H,K-ATPase, since previous work has shown that their complete removal, by peptide N-glycosidase F (PNGase F), greatly increased susceptibility of HKbeta to proteolysis. The possibility of graded protection by different numbers of oligosaccharides was investigated here with the use of mutant HKbeta cDNA, having various N-glycosylation sites mutated (Asn to Gln), transfected into HEK-293 cells. Membrane preparations, two days after transfection, were solubilized in 1% Triton X-100 and subjected to trypsinolysis (pH 8, 37 degrees C, trypsin:protein 1:10-1:25). Relative amounts of HKbeta remaining after 20 min trypsin were determined, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probing of Western blots with an antibody to the HKbeta extracellular domain, by chemiluminescent development of blots and densitometry of resulting films. Maturely glycosylated HKbeta was made significantly more susceptible to trypsin than wild type when at least five oligosaccharides were deleted, while the high-mannose form (pre-beta), from the endoplasmic reticulum, became significantly more susceptible than wild-type pre-beta with removal of only two or more oligosaccharides. For each mutant, and wild type, pre-beta was consistently more susceptible than the mature form. While the number, and kind, of oligosaccharides seem to affect protection for HKbeta against trypsinolysis, other aspects of protein maturation, including proper folding of peptide domains and possible subtle alterations of conformation during Golgi processing, are also likely to contribute to this protection. Copyright 2004 Wiley-VCH Verlag GmbH and Co.

Oligomeric status of the dihydropyridine receptor in aged skeletal muscle.

PubMed

Ryan, M; Carlson, B M; Ohlendieck, K

2000-10-01

A prominent feature of aging is represented by a decrease in muscle mass and strength. Abnormalities in Ca2+ -regulatory membrane complexes are involved in many muscular disorders. In analogy, we determined potential age-related changes in a key component of excitation-contraction coupling, the dihydropyridine receptor. Immunoblotting of the microsomal fraction from aged rabbit muscle revealed a drastic decline in the voltage-sensing alpha1-subunit of this transverse-tubular receptor, but only marginally altered expression of its auxiliary alpha(2)-subunit and the Na+/K+ -ATPase. A shift to slower fibre type characteristics was indicated by an age-related increase in the slow calsequestrin isoform. Chemical crosslinking analysis showed that the triad receptor complex has a comparable tendency of protein-protein interactions in young and aged muscles. Hence, a reduced expression and not modified oligomerization of the principal dihydropyridine receptor subunit might be involved in triggering impaired triadic signal transduction and abnormal Ca2+ -homeostasis resulting in a progressive functional decline of skeletal muscles. Copyright 2001 Academic Press.

Targeting mechanisms of high voltage-activated Ca2+ channels.

PubMed

Herlitze, Stefan; Xie, Mian; Han, Jing; Hümmer, Alexander; Melnik-Martinez, Katya V; Moreno, Rosa L; Mark, Melanie D

2003-12-01

Functional voltage-dependent Ca2+ channel complexes are assembled by three to four subunits: alpha1, beta, alpha2delta subunits (C. Leveque et al., 1994, J. Biol Chem. 269, 6306-6312; M. W. McEnery et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88, 11095-11099) and at least in muscle cells also y subunits (B. M. Curtis and W. A. Catterall, 1984, Biochemistry 23, 2113-2118). Ca2+ channels mediate the voltage-dependent Ca2+ influx in subcellular compartments, triggering such diverse processes as neurotransmitter release, dendritic action potentials, excitation-contraction, and excitation-transcription coupling. The targeting of biophysically defined Ca2+ channel complexes to the correct subcellular structures is, thus, critical to proper cell and physiological functioning. Despite their importance, surprisingly little is known about the targeting mechanisms by which Ca2+ channel complexes are transported to their site of function. Here we summarize what we know about the targeting of Ca2+ channel complexes through the cell to the plasma membrane and subcellular structures.

High energy phosphate transfer by NDPK B/Gbetagammacomplexes--an alternative signaling pathway involved in the regulation of basal cAMP production.

PubMed

Hippe, Hans-Joerg; Wieland, Thomas

2006-08-01

The activation of heterotrimeric G proteins induced by G protein coupled receptors (GPCR) is generally believed to occur by a GDP/GTP exchange at the G protein alpha -subunit. Nevertheless, nucleoside diphosphate kinase (NDPK) and the beta-subunit of G proteins (Gbeta) participate in G protein activation by phosphate transfer reactions leading to the formation of GTP from GDP. Recent work elucidated the role of these reactions. Apparently, the NDPK isoform B (NDPK B) forms a complex with Gbetagamma dimers in which NDPK B acts as a histidine kinase phosphorylating Gbeta at His266. Out of this high energetic phosphoamidate bond the phosphate can be transferred specifically onto GDP. The formed GTP binds to the G protein alpha-subunit and thus activates the respective G protein. Evidence is presented, that this process occurs independent of the classical GPCR-induced GTP/GTP exchange und thus contributes, e.g. to the regulation of basal cAMP synthesis in cells.

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

PubMed Central

Pathare, Ganesh Ramnath; Nagy, István; Bohn, Stefan; Unverdorben, Pia; Hubert, Agnes; Körner, Roman; Nickell, Stephan; Lasker, Keren; Sali, Andrej; Tamura, Tomohiro; Nishioka, Taiki; Förster, Friedrich; Baumeister, Wolfgang; Bracher, Andreas

2012-01-01

Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP. PMID:22187461

A double mutation in exon 6 of the [beta]-hexosaminidase [alpha] subunit in a patient with the B1 variant of Tay-Sachs disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ainsworth, P.J.; Coulter-Mackie, M.B.

1992-10-01

The B1 variant form of Tay-Sachs disease is enzymologically unique in that the causative mutation(s) appear to affect the active site in the [alpha] subunit of [beta]-hexosaminidase A without altering its ability to associate with the [beta] subunit. Most previously reported B1 variant mutations were found in exon 5 within codon 178. The coding sequence of the [alpha] subunit gene of a patient with the B1 variant form was examined with a combination of reverse transcription of mRNA to cDNA, PCR, and dideoxy sequencing. A double mutation in exon 6 has been identified: a G[sub 574][yields]C transversion causing a val[submore » 192][yields]leu change and a G[sub 598][yields] A transition resulting in a val[sub 200][yields]met alteration. The amplified cDNAs were otherwise normal throughout their sequence. The 574 and 598 alterations have been confirmed by amplification directly from genomic DNA from the patient and her mother. Transient-expression studies of the two exon 6 mutations (singly or together) in COS-1 cells show that the G[sub 574][yields]C change is sufficient to cause the loss of enzyme activity. The biochemical phenotype of the 574 alteration in transfection studies is consistent with that expected for a B1 variant mutation. As such, this mutation differs from previously reported B1 variant mutations, all of which occur in exon 5. 31 refs., 2 figs., 2 tabs.« less

Tobacco components stimulate Akt-dependent proliferation and NFkappaB-dependent survival in lung cancer cells.

PubMed

Tsurutani, Junji; Castillo, S Sianna; Brognard, John; Granville, Courtney A; Zhang, Chunyu; Gills, Joell J; Sayyah, Jacqueline; Dennis, Phillip A

2005-07-01

Retrospective studies have shown that patients with tobacco-related cancers who continue to smoke after their diagnoses have lower response rates and shorter median survival compared with patients who stop smoking. To provide insight into the biologic basis for these clinical observations, we tested whether two tobacco components, nicotine or the tobacco-specific carcinogen, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), could activate the Akt pathway and increase lung cancer cell proliferation and survival. Nicotine or NNK, rapidly and potently, activated Akt in non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) cells. Nicotinic activation of Akt increased phosphorylation of multiple downstream substrates of Akt in a time-dependent manner, including GSK-3, FKHR, tuberin, mTOR and S6K1. Since nicotine or NNK bind to cell surface nicotinic acetylcholine receptors (nAchR), we used RT-PCR to assess expression of nine alpha and three beta nAchR subunits in five NSCLC cell lines and two types of primary lung epithelial cells. NSCLC cells express multiple nAchR subunits in a cell line-specific manner. Agonists of alpha3/alpha4 or alpha7 subunits activated Akt in a time-dependent manner, suggesting that tobacco components utilize these subunits to activate Akt. Cellular outcomes after nicotine or NNK administration were also assessed. Nicotine or NNK increased proliferation of NSCLC cells in an Akt-dependent manner that was closely linked with changes in cyclin D1 expression. Despite similar induction of proliferation, only nicotine decreased apoptosis caused by serum deprivation and/or chemotherapy. Protection conferred by nicotine was NFkappaB-dependent. Collectively, these results identify tobacco component-induced, Akt-dependent proliferation and NFkappaB-dependent survival as cellular processes that could underlie the detrimental effects of smoking in cancer patients.

Functional and Structural Impact of Target Uridine Substitutions on the H/ACA Ribonucleoprotein Particle Pseudouridine Synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Jing; Liang, Bo; Li, Hong

2010-09-17

Box H/ACA ribonucleoprotein protein particles catalyze the majority of pseudouridylation in functional RNA. Different from stand alone pseudouridine synthases, the RNP pseudouridine synthase comprises multiple protein subunits and an RNA subunit. Previous studies showed that each subunit, regardless its location, is sensitive to the step of subunit placement at the catalytic center and potentially to the reaction status of the substrate. Here we describe the impact of chemical substitutions of target uridine on enzyme activity and structure. We found that 3-methyluridine in place of uridine inhibited its isomerization while 2{prime}-deoxyuridine or 4-thiouridine did not. Significantly, crystal structures of an archaealmore » box H/ACA RNP bound with the nonreactive and the two postreactive substrate analogues showed only subtle structural changes throughout the assembly except for a conserved tyrosine and a substrate anchoring loop of Cbf5. Our results suggest a potential role of these elements and the subunit that contacts them in substrate binding and product release.« less

Some assembly required: Contributions of Tom Stevens' lab to the V-ATPase field.

PubMed

Graham, Laurie A; Finnigan, Gregory C; Kane, Patricia M

2018-06-01

Tom Stevens' lab has explored the subunit composition and assembly of the yeast V-ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V-ATPase proton pumps and led to the discovery of protein splicing of the V-ATPase catalytic subunit. The Vma - phenotype, characteristic of loss-of-V-ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics. V-ATPase subunit composition proved to be highly conserved among eukaryotes. Genetic screens for new vma mutants led to identification of a set of dedicated V-ATPase assembly factors and helped unravel the complex pathways for V-ATPase assembly. In later years, exploration of the evolutionary history of several V-ATPase subunits provided new information about the enzyme's structure and function. This review highlights V-ATPase work in the Stevens' lab between 1987 and 2017. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular architecture of the TRAPPII complex and implications for vesicle tethering.

PubMed

Yip, Calvin K; Berscheminski, Julia; Walz, Thomas

2010-11-01

Multisubunit tethering complexes participate in the process of vesicle tethering--the initial interaction between transport vesicles and their acceptor compartments. TRAPPII (named for transport protein particle II) is a highly conserved tethering complex that functions in the late Golgi apparatus and consists of all of the subunits of TRAPPI and three additional, specific subunits. We have purified native yeast TRAPPII and characterized its structure and subunit organization by single-particle EM. Our data show that the nine TRAPPII components form a core complex that dimerizes into a three-layered, diamond-shaped structure. The TRAPPI subunits assemble into TRAPPI complexes that form the outer layers. The three TRAPPII-specific subunits cap the ends of TRAPPI and form the middle layer, which is responsible for dimerization. TRAPPII binds the Ypt1 GTPase and probably uses the TRAPPI catalytic core to promote guanine nucleotide exchange. We discuss the implications of the structure of TRAPPII for coat interaction and TRAPPII-associated human pathologies.

Purification of subunits of Escherichia coli DNA gyrase and reconstitution of enzymatic activity.

PubMed

Higgins, N P; Peebles, C L; Sugino, A; Cozzarelli, N R

1978-04-01

Extensively purified DNA gyrase from Escherichia coli is inhibited by nalidixic acid and by novobiocin. The enzyme is composed of two subunits, A and B, which were purified as separate components. Subunit A is the product of the gene controlling sensitivity to nalidixic acid (nalA) because: (i) the electrophoretic mobility of subunit A in the presence of sodium dodecyl sulfate is identical to that of the 105,000-dalton nalA gene product; (ii) mutants that are resistant to nalidixic acid (nalA(r)) produce a drug-resistant subunit A; and (iii) wild-type subunit A confers drug sensitivity to in vitro synthesis of varphiX174 DNA directed by nalA(r) mutants. Subunit B contains a 95,000-dalton polypeptide and is controlled by the gene specifying sensitivity to novobiocin (cou) because cou(r) mutants produce a novobiocin-resistant subunit B and novobiocin-resitant gyrase is made drug sensitive by wild-type subunit B. Subunits A and B associate, so that gyrase was also purified as a complex containing 105,000- and 95,000-dalton polypeptides. This enzyme and gyrase reconstructed from subunits have the same drug sensitivity, K(m) for ATP, and catalytic properties. The same ratio of subunits gives efficient reconstitution of the reactions intrinsic to DNA gyrase, including catalysis of supercoiling of closed duplex DNA, relaxation of supercoiled DNA in the absence of ATP, and site-specific cleavage of DNA induced by sodium dodecyl sulfate.

Application of catalytic adsorptive stripping voltammetry of the cobalt-alpha-benzil dioxime complex to analysis of cobalt traces in metallic zinc.

PubMed

Bobrowski, A

1994-05-01

The catalytic adsorptive stripping voltammetric method with alpha-benzil dioxime and nitrite affords numerous advantages in cobalt determination. The detailed conditions of the determination of the cobalt traces in metallic zinc by catalytic adsorptive stripping voltammetry have been investigated. Both the linear sweep and the differential pulse stripping modes can be used with similar sensitivity. Possible interferences by Mn, Pb, Cu, Ni and Fe are evaluated. In the presence of 5 x 10(5) fold excess of Zn the linear dependence of the cobalt CASV peak current on concentration ranged from 0.05 mug/l to 3 mug/l. Optimal conditions include the accumulation potential of -0.65 V and the accumulation time of 10 sec. The results of the determination of 10(-5)% level of Co in the metallic zinc showed good reproducibility (relative standard deviation, RSD = 0.07) and reliability.

Enhancing photo-catalytic production of organic acids in the cyanobacterium S ynechocystis sp. PCC 6803 Δ glg C , a strain incapable of glycogen storage

DOE PAGES

Carrieri, Damian; Broadbent, Charlie; Carruth, David; ...

2015-01-23

We describe how a key objective in microbial biofuels strain development is to maximize carbon flux to target products while minimizing cell biomass accumulation, such that ideally the algae and bacteria would operate in a photo-catalytic state. A brief period of such a physiological state has recently been demonstrated in the cyanobacterium Synechocystis sp. PCC 6803 ΔglgC strain incapable of glycogen storage. When deprived of nitrogen, the ΔglgC excretes the organic acids alpha-ketoglutarate and pyruvate for a number of days without increasing cell biomass. This study examines the relationship between the growth state and the photo-catalytic state, and characterizes themore » metabolic adaptability of the photo-catalytic state to increasing light intensity. It is found that the culture can transition naturally from the growth state into the photo-catalytic state when provided with limited nitrogen supply during the growth phase. Photosynthetic capacity and pigments are lost over time in the photo-catalytic state. Reversal to growth state is observed with re-addition of nitrogen nutrient, accompanied by restoration of photosynthetic capacity and pigment levels in the cells. While the overall productivity increased under high light conditions, the ratio of alpha-ketoglutarate/pyruvate is altered, suggesting that carbon partition between the two products is adaptable to environmental conditions.« less

Retro-binding thrombin active site inhibitors: identification of an orally active inhibitor of thrombin catalytic activity.

PubMed

Iwanowicz, Edwin J; Kimball, S David; Lin, James; Lau, Wan; Han, W-C; Wang, Tammy C; Roberts, Daniel G M; Schumacher, W A; Ogletree, Martin L; Seiler, Steven M

2002-11-04

A series of retro-binding inhibitors of human alpha-thrombin was prepared to elucidate structure-activity relationships (SAR) and optimize in vivo performance. Compounds 9 and 11, orally active inhibitors of thrombin catalytic activity, were identified to be efficacious in a thrombin-induced lethality model in mice.