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ODS_BOOTSTRAP: assessing the statistical reliability of physical maps by bootstrap resampling.

In the program ODS_BOOTSTRAP we provide a methodology for quickly ordering clones in a genomic library into a physical map and for applying a statistical tool known as the bootstrap to assess the statistical reliability of a clonal ordering. Each clone is assigned a binary fingerprint by one of a variety of experimental approaches to physical mapping. For example, the binary fingerprints might be generated by hybridizing a panel of m probes to a library of n clones. The resulting n x m binary data matrix, X, is input to ODS_BOOTSTRAP, which utilizes the similarity in binary fingerprints of clones to construct a physical map. Under this particular implementation of bootstrap resampling, the m probes (or columns of the data matrix) are sampled randomly with replacement in the computer to generate a new n x m data matrix, X*, from which a second physical map is constructed. The resampling process is repeated 100 or more times to generate 100 or more X* matrices. The resulting 100 or more physical maps are compared with the original physical map based on the original data matrix X by counting how often links in the original physical map reappear. Three confidence statistics are introduced for each link in a physical map. The statistic C1 is defined as the percentage of time two neighboring clones on the original map reappear as neighbors under resampling. The statistic C2 is defined as the percentage of time that two neighboring clones i and j on the original map reappear as neighbors or that a clone with an identical binary fingerprint to clone i reappears as a neighbor to clone j. The statistic C3 is defined as the percentage of time that two neighboring clones on the original map reappear in the same contig under resampling.

Algorithms↗

Taxonomy workbench.

UNLABELLED: At advanced stages of working with user-defined protein and gene sequence collections, it is frequently necessary to link these data to the taxonomic tree and to extract subsets in accordance with taxonomic considerations. Since no general automatic tools had been available, this was a tedious manual effort. Our taxonomy workbench allows processing of sequence sets, mapping of these sets onto the taxonomic tree, collection of taxonomic subsets from them and printing of the whole tree or some part of it. As a side effect, the system enables queries to and navigation within the taxonomy database. AVAILABILITY: An implementation of the taxonomy workbench is accessible for public use as a www-service at http://mendel.imp.univie.ac.at/taxonomy/. Software components for the command-line and for the www-version are available on request. CONTACT: Georg.Schneider@nt.imp.univie.ac.at; Frank.Eisenhaber@nt.imp.univie.ac.at SUPPLEMENTARY INFORMATION: Documentation for the taxonomy workbench can be accessed at http://mendel.imp.univie.ac.at/taxonomy/help.html.

Databases, Nucleic Acid↗

Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing.

Su(var)3-9 is a dominant modifier of heterochromatin-induced gene silencing. Like its mammalian and Schizosaccharomyces pombe homologues, Su(var) 3-9 encodes a histone methyltransferase (HMTase), which selectively methylates histone H3 at lysine 9 (H3-K9). In Su(var)3-9 null mutants, H3-K9 methylation at chromocentre heterochromatin is strongly reduced, indicating that SU(VAR)3-9 is the major heterochromatin-specific HMTase in Drosophila. SU (VAR)3-9 interacts with the heterochromatin-associated HP1 protein and with another silencing factor, SU(VAR)3-7. Notably, SU(VAR)3-9-HP1 interaction is interdependent and governs distinct localization patterns of both proteins. In Su(var)3-9 null mutants, concentration of HP1 at the chromocentre is nearly lost without affecting HP1 accumulation at the fourth chromosome. By contrast, in HP1 null mutants SU(VAR)3-9 is no longer restricted at heterochromatin but broadly dispersed across the chromosomes. Despite this interdependence, Su(var)3-9 dominates the PEV modifier effects of HP1 and Su(var)3-7 and is also epistatic to the Y chromosome effect on PEV. Finally, the human SUV39H1 gene is able to partially rescue Su(var)3-9 silencing defects. Together, these data indicate a central role for the SU(VAR)3-9 HMTase in heterochromatin-induced gene silencing in Drosophila.

Animals↗

Involvement of Fes/Fps tyrosine kinase in semaphorin3A signaling.

Collapsin response mediator proteins (CRMPs)/TOAD64/Ulips/DRPs and CRAM have emerged as strong candidates for a role in semaphorin signaling. In this study we identified Fes/Fps (Fes) tyrosine kinase in the CRMP-CRAM complex and investigated whether Fes was involved in semaphorin3A (Sema3A) signaling. In COS-7 cells, the interaction between Fes and plexinA1 (PlexA1) and the tyrosine phosphorylation of PlexA1 by Fes were observed; however, these events were significantly attenuated by co-expression of neuropilin-1 (NP-1). Even with NP-1 co-expression, Sema3A was able to enhance the association of Fes with PlexA1 and Fes-mediated tyrosine phosphorylation of PlexA1, CRAM and CRMP2. Co-expression of Fes with PlexA1 exhibited COS-7 cell contraction activity, indicating that Fes can convert inactive PlexA1 to its active form, whereas combination of Fes/NP-1/PlexA1 or Fes kinase-negative mutants/PlexA1 did not alter cell morphology. Finally, Sema3A-induced growth cone collapse of dorsal root ganglion neurons was suppressed by expression of Fes kinase-negative mutants. Taken together, our findings suggest that Fes links Sema3A signals to CRMP-CRAM, and that NP-1 negatively regulates PlexA1 activation by Fes in resting condition.

Adaptor Proteins, Signal Transducing↗

FXYD7 is a brain-specific regulator of Na,K-ATPase alpha 1-beta isozymes.

Recently, corticosteroid hormone-induced factor (CHIF) and the gamma-subunit, two members of the FXYD family of small proteins, have been identified as regulators of renal Na,K-ATPase. In this study, we have investigated the tissue distribution and the structural and functional properties of FXYD7, another family member which has not yet been characterized. Expressed exclusively in the brain, FXYD7 is a type I membrane protein bearing N-terminal, post-translationally added modifications on threonine residues, most probably O-glycosylations that are important for protein stabilization. Expressed in Xenopus oocytes, FXYD7 can interact with Na,K-ATPase alpha 1-beta 1, alpha 2-beta 1 and alpha 3-beta 1 but not with alpha-beta 2 isozymes, whereas, in brain, it is only associated with alpha 1-beta isozymes. FXYD7 decreases the apparent K(+) affinity of alpha 1-beta 1 and alpha 2-beta 1, but not of alpha 3-beta1 isozymes. These data suggest that FXYD7 is a novel, tissue- and isoform-specific Na,K-ATPase regulator which could play an important role in neuronal excitability.

Action Potentials↗

Phosphorylation-dependent ubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3 ligase.

The androgen receptor (AR) controls several biological functions including prostate cell growth and apoptosis. However, the mechanism by which AR maintains its stability to function properly remains largely unknown. Here we show that Akt and Mdm2 form a complex with AR and promote phosphorylation-dependent AR ubiquitylation, resulting in AR degradation by the proteasome. The effect of Akt on AR ubiquitylation and degradation is markedly impaired in a Mdm2-null cell line compared with the wild-type cell line, suggesting that Mdm2 is involved in Akt-mediated AR ubiquitylation and degradation. Furthermore, we demonstrate that the E3 ligase activity of Mdm2 and phosphorylation of Mdm2 by Akt are essential for Mdm2 to affect AR ubiquitylation and degradation. These results suggest that phosphorylation-dependent AR ubiquitylation and degradation by Akt require the involvement of Mdm2 E3 ligase activity, a novel mechanism that provides insight into how AR is targeted for degradation.

Animals↗

Ca2+-dependent block of CREB-CBP transcription by repressor DREAM.

The calcium-binding protein DREAM binds specifically to DRE sites in the DNA and represses transcription of target genes. Derepression at DRE sites following PKA activation depends on a specific interaction between alphaCREM and DREAM. Two leucine-charged residue-rich domains (LCD) located in the kinase-inducible domain (KID) and in the leucine zipper of alphaCREM and two LCDs in DREAM participate in a two-site interaction that results in the loss of DREAM binding to DRE sites and derepression. Since the LCD motif located within the KID in CREM is also present in CREB, and maps in a region critical for the recruitment of CBP, we investigated whether DREAM may affect CRE-dependent transcription. Here we show that in the absence of Ca2+ DREAM binds to the LCD in the KID of CREB. As a result, DREAM impairs recruitment of CBP by phospho CREB and blocks CBP-mediated transactivation at CRE sites in a Ca2+-dependent manner. Thus, Ca2+-dependent interactions between DREAM and CREB represent a novel point of cross-talk between cAMP and Ca2+ signalling pathways in the nucleus.

Brain↗

The chromatin remodeling complex NoRC targets HDAC1 to the ribosomal gene promoter and represses RNA polymerase I transcription.

Mammalian chromatin remodeling complexes are involved in both activation and repression of transcription. Here, we show that NoRC, a SNF2h- containing nucleolar chromatin remodeling complex, represses ribosomal gene transcription. NoRC-mediated rDNA silencing was alleviated by trichostatin A, indicating that histone deacetylation is causally involved in silencing. Chromatin immunoprecipitation experiments demonstrate that overexpression of TIP5, the large subunit of NoRC, mediates deacetylation of nucleosomes in the vicinity of the rDNA promoter. Protein-protein interaction assays reveal association of TIP5 with the histone deacetylase HDAC1 in vivo and in vitro. Deletion of the C-terminal PHD finger and bromodomain abolishes the interaction of TIP5 and HDAC1, and abrogates transcriptional repression. The results suggest that NoRC silences the rDNA locus by targeting the SIN3 corepressor complex to the rDNA promoter, thereby establishing a repressed chromatin structure.

3T3 Cells↗

Hemoglobin binds melanoma cell tissue factor and enhances its procoagulant activity.

Tissue factor (TF), the membrane-bound glycoprotein that normally initiates the coagulation pathway, is expressed on the surface of various cells including endothelial cells, fibroblasts, monocytes and tumor cells. We recently reported that hemoglobin (Hb) enhances TF expression and procoagulant activity on TF-bearing human A375 malignant melanoma cells. To elucidate the mechanism of Hb-induced TF expression, we studied the interaction between purified TF from human A375 malignant melanoma cells and Hb. Selective binding of highly purified melanoma cell TF-apoprotein to Hb was demonstrated under native conditions using a dot-immunobinding assay and under denaturing conditions by Western blotting. The complex formation between purified melanoma cell TF-apoprotein and Hb was also demonstrated by the binding of fluid-phase Hb to immobilized TF-apoprotein (0-2.0 microg/ml) in an enzyme-linked immunosorbent assay. The binding was specific, concentration-dependent, saturable and inhibited significantly (60%) by Concanavalin-A. Hb enhanced the factor X-activating procoagulant activity of melanoma cell TF in a concentration-dependent manner, but had no effect on recombinant human TF. Concanavalin-A and wheat germ agglutinin significantly (60%) inhibited the Hb-induced procoagulant activity of malignant cell TF. We conclude that TF-apoprotein selectively binds Hb, most probably via the carbohydrate moieties (alpha-d-glucosyl; alpha-d-mannosyl and N-acetyl-beta-d-glucosaminyl residues) of TF, and enhances its procoagulant activity. The physiological significance of this interaction remains to be established.

Apoproteins↗

Infusing teaching thinking skills into subject-area instruction.

Students can be taught clinical reasoning actively with the adaptation of the infusion approach for "teaching of" thinking. The authors demonstrate the "teaching of" critical thinking strategies for diagnostic decisions. The instructor uses a thinking map and models the process of diagnosis step by step. Working in small groups, students practice, monitor, and revise their strategies of clinical reasoning.

Algorithms↗

Membrane orientation and oligomerization of the small hydrophobic protein of human respiratory syncytial virus.

Previous work has demonstrated that the small hydrophobic (SH) protein of human respiratory syncytial virus (RSV) A2 strain is a 64 amino acid integral membrane protein that accumulates intracellularly as an unglycosylated major species (SH0), a minor species truncated at the amino terminus and two N-glycosylated species one of which contains a further addition of polylactosamine. In this study, the membrane orientation of SH0 was mapped by trypsinization of intact RSV-infected cells followed by washout, lysis and immunoprecipitation of protected fragments with antisera specific for the protein termini. This showed that the C terminus is extracellular and the SH protein was not detectably palmitylated. Analysis of the SH protein by sedimentation on sucrose gradients showed that it rapidly assembles into a homo-oligomer that co-sediments with the F protein tetramer. Interestingly, all forms of the SH protein were found in the oligomeric fraction. Chemical cross-linking generated species which appeared to represent dimers, trimers, tetramers and pentamers as well as a minor species of 180K which might correspond to the oligomeric form detected by sucrose gradient sedimentation.

Animals↗

Smad3 allostery links TGF-beta receptor kinase activation to transcriptional control.

Smad3 transduces the signals of TGF-betas, coupling transmembrane receptor kinase activation to transcriptional control. The membrane-associated molecule SARA (Smad Anchor for Receptor Activation) recruits Smad3 for phosphorylation by the receptor kinase. Upon phosphorylation, Smad3 dissociates from SARA and enters the nucleus, in which its transcriptional activity can be repressed by Ski. Here, we show that SARA and Ski recognize specifically the monomeric and trimeric forms of Smad3, respectively. Thus, trimerization of Smad3, induced by phosphorylation, simultaneously activates the TGF-beta signal by driving Smad3 dissociation from SARA and sets up the negative feedback mechanism by Ski. Structural models of the Smad3/SARA/receptor kinase complex and Smad3/Ski complex provide insights into the molecular basis of regulation.

Allosteric Regulation↗

Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U.

beta-TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor subunit of the SCF(beta-TrCP) ubiquitin ligase (E3). Surprisingly, although its two recognized substrates, IkappaB(alpha) and beta-catenin, are present in the cytoplasm, we have found that E3RS is located predominantly in the nucleus. Here we report the isolation of the major E3RS-associated protein, hnRNP-U, an abundant nuclear phosphoprotein. This protein occupies E3RS in a specific and stoichiometric manner, stabilizes the E3 component, and is likely responsible for its nuclear localization. hnRNP-U binding was abolished by competition with a pIkappaB(alpha) peptide, or by a specific point mutation in the E3RS WD region, indicating an E3-substrate-type interaction. However, unlike pI(kappa)Balpha, which is targeted by SCF(beta-TrCP) for degradation, the E3-bound hnRNP-U is stable and is, therefore, a pseudosubstrate. Consequently, hnRNP-U engages a highly neddylated active SCF(beta-TrCP), which dissociates in the presence of a high-affinity substrate, resulting in ubiquitination of the latter. Our study points to a novel regulatory mechanism, which secures the localization, stability, substrate binding threshold, and efficacy of a specific protein-ubiquitin ligase.

Active Transport, Cell Nucleus↗

Coarsening of sand ripples in mass transfer models.

Coarsening of sand ripples is studied in a one-dimensional stochastic model, where neighboring ripples exchange mass with algebraic rates, Gamma(m) approximately m(gamma), and ripples of zero mass are removed from the system. For gamma<0, ripples vanish through rare fluctuations and the average ripple mass grows as (t) approximately -gamma(-1)ln(t). Temporal correlations decay as t(-1/2) or t(-2/3) depending on the symmetry of the mass transfer, and asymptotically the system is characterized by a product measure. The stationary ripple mass distribution is obtained exactly. For gamma>0, ripple evolution is linearly unstable, and the noise in the dynamics is irrelevant. For gamma=1, the problem is solved on the mean-field level, but the mean-field theory does not adequately describe the full behavior of the coarsening. In particular, it fails to account for the numerically observed universality with respect to the initial ripple size distribution. The results are not restricted to sand ripple evolution since the model can be mapped to zero range processes, urn models, exclusion processes, and cluster-cluster aggregation.

Journal Article↗

Frequency of elemental events of intracellular Ca(2+) dynamics.

The dynamics of intracellular Ca(2+) is driven by random events called Ca(2+) puffs, in which Ca(2+) is liberated from intracellular stores. We show that the emergence of Ca(2+) puffs can be mapped to an escape process. The mean first passage times that correspond to the stochastic fraction of puff periods are computed from a novel master equation and two Fokker-Planck equations. Our results demonstrate that the mathematical modeling of Ca(2+) puffs has to account for the discrete character of the Ca(2+) release sites and does not permit a continuous description of the number of open channels.

Calcium↗

Identification of post-translational modifications via blind search of mass-spectra.

Post-translational modifications (PTMs) are of great biological importance. Most existing approaches perform a restrictive search that can only take into account a few types of PTMs and ignore all others. We describe an unrestrictive PTM search algorithm that searches for all types of PTMs at once in a blind mode, i.e., without knowing which PTMs exist in a sample. The blind PTM identification opens a possibility to study the extent and frequencies of different types of PTMs, still an open problem in proteomics. Using our new algorithm, we were able to construct a two-dimensional PTM frequency matrix that reflects the number of MS/MS spectra in a sample for each putative PTM type and each amino acid. Application of this approach to a large IKKb dataset resulted in the largest set of PTMs reported for a single MS/MS sample so far. We demonstrate an excellent correlation between high values in the PTM frequency matrix and known PTMs thus validating our approach. We further argue that the PTM frequency matrix may reveal some still unknown modifications that warrant further experimental validation.

Algorithms↗

Introduction to positional cloning.

The most common procedure for isolating genes that predispose to a certain disease is positional cloning. This process is initiated by mapping the responsible gene to its location on a chromosome linked to the disease. Successive cloning of the region results in the isolation of candidate genes. Finally, identification of mutations by mutational analysis points to the correct gene.

Chromosome Mapping↗

Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected Sf21 cells. Characterization of polypeptides and posttranslational modifications.

Human interleukin 2 (IL-2) and human IL-2 mutant proteins, with artificially introduced N-glycosylation or O-glycosylation sites, have been expressed in a lepidopteran cell line (Sf21, Spodoptera frugiperda) using recombinant baculovirus vectors. Only approximately 25% of the total recombinant IL-2 protein synthesized by Sf21 cells was secreted into the culture medium. Significant N-terminal truncations were detected in the secreted polypeptides (up to 85% of the molecules). Alanine and proline were absent in the major truncated forms; the first 3-5 amino acids were also absent in a small proportion of the purified proteins. The introduction of potential artificial O-glycosylation peptide sequences (..GGKAPTPPPK..), to the C-terminus or between positions 80 and 81 of the IL-2 polypeptide chain, resulted in the secretion of unglycosylated and O-glycosylated variant forms. Fast atom bombardment mass spectrometry, compositional analysis and methylation analysis, of the tryptic glycopeptide APTPPPK, revealed the presence of either GalNAc or the disaccharide Gal(beta 1-3)GalNAc as the only carbohydrate constituents attached exclusively to Thr in this peptide, in a specific ratio for each individual IL-2 mutant protein. The Gal(beta 1-3)GalNAc protein forms could be partially altered in vitro to mammalian-type glycoforms by porcine liver beta-galactoside alpha-2,3-sialyltransferase in the presence of CMP-N-acetylneuraminic acid. An IL-2 mutant form, with an 11-amino-acid peptide of human interferon-beta at position 4, which includes its only N-glycosylation site, had exclusively truncated proximally fucosylated oligomannosidic glycans; Man3GlcNAc[Fuc(alpha 1-6)]GlcNAc or Man2GlcNAc[Fuc(alpha 1-6)]GlcNAc structures, in a ratio of 3:1, were detected in the secreted proteins. No evidence was obtained for the presence of secreted proteins with complex oligosaccharide chains, irrespective of the cell-culture conditions used or the harvesting time, for infected cells with recombinant baculovirus constructs.

Amino Acid Sequence↗