BIND—The Biomolecular Interaction Network Database

The Biomolecular Interaction Network Database (BIND; http://binddb.org) is a database designed to store full descriptions of interactions, molecular complexes and pathways. Development of the BIND 2.0 data model has led to the incorporation of virtually all components of molecular mechanisms including interactions between any two molecules composed of proteins, nucleic acids and small molecules. Chemical reactions, photochemical activation and conformational changes can also be described. Everything from small molecule biochemistry to signal transduction is abstracted in such a way that graph theory methods may be applied for data mining. The database can be used to study networks of interactions, to map pathways across taxonomic branches and to generate information for kinetic simulations. BIND anticipates the coming large influx of interaction information from high-throughput proteomics efforts including detailed information about post-translational modifications from mass spectrometry. Version 2.0 of the BIND data model is discussed as well as implementation, content and the open nature of the BIND project. The BIND data specification is available as ASN.1 and XML DTD.

The RESID Database of protein structure modifications and the NRL-3D Sequence–Structure Database

The RESID Database is a comprehensive collection of annotations and structures for protein post-translational modifications including N-terminal, C-terminal and peptide chain cross-link modifications. The RESID Database includes systematic and frequently observed alternate names, Chemical Abstracts Service registry numbers, atomic formulas and weights, enzyme activities, taxonomic range, keywords, literature citations with database cross-references, structural diagrams and molecular models. The NRL-3D Sequence–Structure Database is derived from the three-dimensional structure of proteins deposited with the Research Collaboratory for Structural Bioinformatics Protein Data Bank. The NRL-3D Database includes standardized and frequently observed alternate names, sources, keywords, literature citations, experimental conditions and searchable sequences from model coordinates. These databases are freely accessible through the National Cancer Institute–Frederick Advanced Biomedical Computing Center at these web sites: http://www.ncifcrf.gov/RESID, http://www.ncifcrf.gov/ NRL-3D; or at these National Biomedical Research Foundation Protein Information Resource web sites: http://pir.georgetown.edu/pirwww/dbinfo/resid.html, http://pir.georgetown.edu/pirwww/dbinfo/nrl3d.html

iProClass: an integrated, comprehensive and annotated protein classification database

The iProClass database is an integrated resource that provides comprehensive family relationships and structural and functional features of proteins, with rich links to various databases. It is extended from ProClass, a protein family database that integrates PIR superfamilies and PROSITE motifs. The iProClass currently consists of more than 200 000 non-redundant PIR and SWISS-PROT proteins organized with more than 28 000 superfamilies, 2600 domains, 1300 motifs, 280 post-translational modification sites and links to more than 30 databases of protein families, structures, functions, genes, genomes, literature and taxonomy. Protein and family summary reports provide rich annotations, including membership information with length, taxonomy and keyword statistics, full family relationships, comprehensive enzyme and PDB cross-references and graphical feature display. The database facilitates classification-driven annotation for protein sequence databases and complete genomes, and supports structural and functional genomic research. The iProClass is implemented in Oracle 8i object-relational system and available for sequence search and report retrieval at http://pir.georgetow n.edu/iproclass/.

The InterPro database, an integrated documentation resource for protein families, domains and functional sites

Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1 000 000 hits from 462 500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk.

Long-term potentiation increases tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit 2B in rat dentate gyrus in vivo.

Long-term potentiation (LTP) is a form of synaptic memory that may subserve developmental and behavioral plasticity. An intensively investigated form of LTP is dependent upon N-methyl-D-aspartate (NMDA) receptors and can be elicited in the dentate gyrus and hippocampal CA1. Induction of this type of LTP is triggered by influx of Ca2+ through activated NMDA receptors, but the downstream mechanisms of induction, and even more so of LTP maintenance, remain controversial. It has been reported that the function of NMDA receptor channel can be regulated by protein tyrosine kinases and protein phosphatases and that inhibition of protein tyrosine kinases impairs induction of LTP. Herein we report that LTP in the dentate gyrus is specifically correlated with tyrosine phosphorylation of the NMDA receptor subunit 2B in an NMDA receptor-dependent manner. The effect is observed with a delay of several minutes after LTP induction and persists in vivo for several hours. The potential relevance of this post-translational modification to mechanisms of LTP and circuit plasticity is discussed.

CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency.

Tumor necrosis factor (TNF) mediates a wide variety of disease states including septic shock, acute and chronic inflammation, and cachexia. Recently, a multivalent guanylhydrazone (CNI-1493) developed as an inhibitor of macrophage activation was shown to suppress TNF production and protect against tissue inflammation and endotoxin lethality [Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M., Zimmerman, G. A., Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., Manogue, K. R., Tortolani, A. J., Cerami, A. & Tracey, K. J. (1995) Mol. Med. 1, 254-266, and Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Zhang, X., Bukrinsky, M., Ulrich, P., Cerami, A. & Tracey, J. (1996) J. Exp. Med., in press]. We have now elucidated the mechanism by which CNI-1493 inhibits macrophage TNF synthesis and show here that it acts through suppression of TNF translation efficiency. CNI-1493 blocked neither the lipopolysaccharide (LPS)-induced increases in the expression of TNF mRNA nor the translocation of nuclear factor NF-kappa B to the nucleus in macrophages activated by 15 min of LPS stimulation, indicating that CNI-1493 does not interfere with early NF-kappa B-mediated transcriptional regulation of TNF. However, synthesis of the 26-kDa membrane form of TNF was effectively blocked by CNI-1493. Further evidence for the translational suppression of TNF is given by experiments using chloram-phenicol acetyltransferase (CAT) constructs containing elements of the TNF gene that are involved in TNF translational regulation. Both the 5' and 3' untranslated regions of the TNF gene were required to elicit maximal translational suppression by CNI-1493. Identification of the molecular target through which CNI-1493 inhibits TNF translation should provide insight into the regulation of macrophage activation and mechanisms of inflammation.

Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.

Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

Transgenic mice and sheep secrete only low levels of human factor IX in their milk because of an aberrant splicing of the transgene RNA in the mammary gland. Removal of the cryptic 3' splice site prevents this splicing and leads to the production of relatively high levels of factor IX. The purified protein is fully active showing that the mammary gland is capable of the efficient post-translational modification of this protein and that transgenic animals are a suitable means of its production.

Proteolysis of major histocompatibility complex class II-associated invariant chain is regulated by the alternatively spliced gene product, p41.

Invariant chain (Ii) is an intracellular type II transmembrane glycoprotein that is associated with major histocompatibility complex class II molecules during biosynthesis. Ii exists in two alternatively spliced forms, p31 and p41. Both p31 and p41 facilitate folding of class II molecules, promote egress from the endoplasmic reticulum, prevent premature peptide binding, and enhance localization to proteolytic endosomal compartments that are thought to be the sites for Ii degradation, antigen processing, and class II-peptide association. In spite of the dramatic and apparently equivalent effects that p31 and p41 have on class II biosynthesis, the ability of invariant chain to enhance antigen presentation to T cells is mostly restricted to p41. Here we show that degradation of Ii leads to the generation of a 12-kDa amino-terminal fragment that in p41-positive, but not in p31-positive, cells remains associated with class II molecules for an extended time. Interestingly, we find that coexpression of the two isoforms results in a change in the pattern of p31 degradation such that endosomal processing of p31 also leads to extended association of a similar 12-kDa fragment with class II molecules. These data raise the possibility that p41 may have the ability to impart its pattern of proteolytic processing on p31 molecules expressed in the same cells. This would enable a small number of p41 molecules to modify the post-translational transport and/or processing of an entire cohort of class II-Ii complexes in a manner that could account for the unique ability of p41 to enhance antigen presentation.

Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA

Several mutations that cause severe forms of the human disease autosomal dominant retinitis pigmentosa cluster in the C-terminal region of rhodopsin. Recent studies have implicated the C-terminal domain of rhodopsin in its trafficking on specialized post-Golgi membranes to the rod outer segment of the photoreceptor cell. Here we used synthetic peptides as competitive inhibitors of rhodopsin trafficking in the frog retinal cell-free system to delineate the potential regulatory sequence within the C terminus of rhodopsin and model the effects of severe retinitis pigmentosa alleles on rhodopsin sorting. The rhodopsin C-terminal sequence QVS(A)PA is highly conserved among different species. Peptides that correspond to the C terminus of bovine (amino acids 324–348) and frog (amino acids 330–354) rhodopsin inhibited post-Golgi trafficking by 50% and 60%, respectively, and arrested newly synthesized rhodopsin in the trans-Golgi network. Peptides corresponding to the cytoplasmic loops of rhodopsin and other control peptides had no effect. When three naturally occurring mutations: Q344ter (lacking the last five amino acids QVAPA), V345M, and P347S were introduced into the frog C-terminal peptide, the inhibitory activity of the peptides was no longer detectable. These observations suggest that the amino acids QVS(A)PA comprise a signal that is recognized by specific factors in the trans-Golgi network. A lack of recognition of this sequence, because of mutations in the last five amino acids causing autosomal dominant retinitis pigmentosa, most likely results in abnormal post-Golgi membrane formation and in an aberrant subcellular localization of rhodopsin.

Calcium regulation of a slow post-spike hyperpolarization in vagal afferent neurons

Activation of distinct classes of potassium channels can dramatically affect the frequency and the pattern of neuronal firing. In a subpopulation of vagal afferent neurons (nodose ganglion neurons), the pattern of impulse activity is effectively modulated by a Ca2+-dependent K+ current. This current produces a post-spike hyperpolarization (AHPslow) that plays a critical role in the regulation of membrane excitability and is responsible for spike-frequency accommodation in these neurons. Inhibition of the AHPslow by a number of endogenous autacoids (e.g., histamine, serotonin, prostanoids, and bradykinin) results in an increase in the firing frequency of vagal afferent neurons from <0.1 to >10 Hz. After a single action potential, the AHPslow in nodose neurons displays a slow rise time to peak (0.3–0.5 s) and a long duration (3–15 s). The slow kinetics of the AHPslow are due, in part, to Ca2+ discharge from an intracellular Ca2+-induced Ca2+ release (CICR) pool. Action potential-evoked Ca2+ influx via either L or N type Ca2+ channels triggers CICR. Surprisingly, although L type channels generate 60% of action potential-induced CICR, only Ca2+ influx through N type Ca2+ channels can trigger the CICR-dependent AHPslow. These observations suggest that a close physical proximity exists between endoplasmic reticulum ryanodine receptors and plasma membrane N type Ca2+ channels and AHPslow potassium channels. Such an anatomical relation might be particularly beneficial for modulation of spike-frequency adaptation in vagal afferent neurons.

cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.

Regulation of p53 expression by thymidylate synthase

Previous studies showed that thymidylate synthase (TS), as an RNA binding protein, regulates its own synthesis by impairing the translation of TS mRNA. In this report, we present evidence that p53 expression is affected in a similar manner by TS. For these studies, we used a TS-depleted human colon cancer HCT-C cell that had been transfected with either the human TS cDNA or the Escherichia coli TS gene. The level of p53 protein in transfected cells overexpressing human TS was significantly reduced when compared with its corresponding parent HCT-C cells. This suppression of p53 expression was the direct result of decreased translational efficiency of p53 mRNA. Similar results were obtained upon transfection of HCT-C cells with pcDNA 3.1 (+) containing the E. coli TS gene. These findings provide evidence that TS, from diverse species, specifically regulates p53 expression at the translational level. In addition, TS-overexpressing cells with suppressed levels of p53 are significantly impaired in their ability to arrest in G1 phase in response to exposure to a DNA-damaging agent such as γ-irradiation. These studies provide support for the in vivo biological relevance of the interaction between TS and p53 mRNA and identify a molecular pathway for controlling p53 expression.

Induction of apoptosis in rhabdomyosarcoma cells through down-regulation of PAX proteins

The expression of a number of human paired box-containing (PAX) genes has been correlated with various types of tumors. Novel fusion genes encoding chimeric fusion proteins have been found in the pediatric malignant tumor alveolar rhabdomyosarcoma (RMS). They are generated by two chromosomal translocations t(2;13) and t(1;13) juxtaposing PAX3 or PAX7, respectively, with a forkhead domain gene FKHR. Here we describe that specific down-regulation of the t(2;13) translocation product in alveolar RMS cells by antisense oligonucleotides results in reduced cellular viability. Cells of embryonal RMS, the other major histiotype of this tumor, were found to express either wild type PAX3 or PAX7 at elevated levels when compared with primary human myoblasts. Treatment of corresponding embryonal RMS cells with antisense olignucleotides directed against the mRNA translational start site of either one of these two transcription factors similarly triggers cell death, which is most likely due to induction of apoptosis. Retroviral mediated ectopic expression of mouse Pax3 in a PAX7 expressing embryonal RMS cell line could partially rescue antisense induced apoptosis. These data suggest that the PAX3/FKHR fusion gene and wild-type PAX genes play a causative role in the formation of RMS and presumably other tumor types, possibly by suppressing the apoptotic program that would normally eliminate these cells.

Rapid down-regulation of mammalian Period genes during behavioral resetting of the circadian clock

The pervasive role of circadian clocks in regulating physiology and behavior is widely recognized. Their adaptive value is their ability to be entrained by environmental cues such that the internal circadian phase is a reliable predictor of solar time. In mammals, both light and nonphotic behavioral cues can entrain the principal oscillator of the hypothalamic suprachiasmatic nuclei (SCN). However, although light can advance or delay the clock during circadian night, behavioral events trigger phase advances during the subjective day, when the clock is insensitive to light. The recent identification of Period (Per) genes in mammals, homologues of dperiod, which encodes a core element of the circadian clockwork in Drosophila, now provides the opportunity to explain circadian timing and entrainment at a molecular level. In mice, expression of mPer1 and mPer2 in the SCN is rhythmic and acutely up-regulated by light. Moreover, the temporal relations between mRNA and protein cycles are consistent with a clock based on a transcriptional/translational feedback loop. Here we describe circadian oscillations of Per1 and Per2 in the SCN of the Syrian hamster, showing that PER1 protein and mRNA cycles again behave in a manner consistent with a negative-feedback oscillator. Furthermore, we demonstrate that nonphotic resetting has the opposite effect to light: acutely down-regulating these genes. Their sensitivity to nonphotic resetting cues supports their proposed role as core elements of the circadian oscillator. Moreover, this study provides an explanation at the molecular level for the contrasting but convergent effects of photic and nonphotic cues on the clock.