The fic domain: regulation of cell signaling by adenylylation.

We show that the secreted antigen, IbpA, of the respiratory pathogen Histophilus somni induces cytotoxicity in mammalian cells via its Fic domains. Fic domains are defined by a core HPFxxGNGR motif and are conserved from bacteria to humans. We demonstrate that the Fic domains of IbpA catalyze a unique reversible adenylylation event that uses ATP to add an adenosine monophosphate (AMP) moiety to a conserved tyrosine residue in the switch I region of Rho GTPases. This modification requires the conserved histidine of the Fic core motif and renders Rho GTPases inactive. We further demonstrate that the only human protein containing a Fic domain, huntingtin yeast-interacting protein E (HYPE), also adenylylates Rho GTPases in vitro. Thus, we classify Fic domain-containing proteins as a class of enzymes that mediate bacterial pathogenesis as well as a previously unrecognized eukaryotic posttranslational modification that may regulate key signaling events.

RNA. Introduction.

Two scientists walk into a bar. After a pint and an exchange of pleasantries, one says to the other, "Where do you come from? Scientifically, I mean." The queried scientist responds, "Out of the RNA world." "Don't we all," the asker responds chuckling. Fifteen years ago, the joke would have been made with a nod to the notion that life arose from an RNA-based precursor, the so-called "RNA world." Yet had this conversation happened last week, the scientists would also be grinning in appreciation of the extent to which contemporary cellular biology is steeped in all things RNA. Ours is truly an RNA world.In this year's special review issue, the Cell editorial team has brought together articles focused on RNA in the modern world, providing perspectives on classical and emerging areas of inquiry. We extend our thanks to the many distinguished experts who contributed their time and effort as authors and reviewers to make the issue informative, thought-provoking, and timely. We hope that this collection of articles, written as we stand on the verge of a new wave of RNA biology, edifies and inspires by revealing the inner workings of these versatile molecules and by highlighting the next key questions that need to be addressed as we strive to understand the full functional scope of RNA in cells.

Protection of mice against H. somni septicemia by vaccination with recombinant immunoglobulin binding protein subunits.

Histophilus somni causes bovine pneumonia as well as septicemia and its sequelae but mechanisms of virulence and protective immunity are poorly understood. Since surface immunoglobulin binding proteins are virulence factors, we addressed their role as protective antigens in a mouse model of H. somni septicemia. Immunoglobulin binding protein A (IbpA), has homology to Bordetella pertussis filamentous hemagglutinin and other large bacterial exoproteins. IbpA is a major surface antigen encoded by the ibpA gene with many domains that may be important in pathogenesis and immune protection. Three IbpA recombinant protein subunits, IbpA3, IbpA5 and IbpADR2 were chosen for study because of putative functional domains and motifs. These recombinant GST fusion subunit proteins were compared with GST (negative control), formalin-killed H. somni (commercial vaccine control), live H. somni (to induce convalescent immunity) and H. somni culture supernatant (containing IbpA shed from the bacterial surface). In vaccination/challenge studies, both live H. somni (convalescent immunity) and supernatant protected equally but formalin-killed H. somni and GST did not protect against septicemia. The DR2 and A3 subunits protected moderately well and induced antibody responses against supernatant antigen and the homologous subunit in ELISA but not against whole cell antigens. Supernatant immunization protected better than the IbpA subunit antigens and induced high antibody activity against both whole cells and supernatant antigens. The results indicate that culture supernatant antigens or perhaps recombinant IbpA subunits may be useful in H. somni vaccines. These studies also provide insight into the contribution of IbpA domains to pathogenesis of H. somni septicemia.

Semaphorins command cells to move.

Semaphorins are secreted or transmembrane proteins that regulate cell motility and attachment in axon guidance, vascular growth, immune cell regulation and tumour progression. The main receptors for semaphorins are plexins, which have established roles in regulating Rho-family GTPases. Recent work shows that plexins can also influence R-Ras, which, in turn, can regulate integrins. Such regulation is probably a common feature of semaphorin signalling and contributes substantially to our understanding of semaphorin biology.

Mapping netrin receptor binding reveals domains of Unc5 regulating its tyrosine phosphorylation.

Netrin and its receptors Unc5 and deleted in colorectal carcinoma (DCC) regulate axon guidance and cell migration. We defined domains involved in the interactions between netrin-1, DCC, and Unc5c. We show that Unc5 requires both Ig domains to interact with netrin. DCC binds through the fourth fibronectin type III domain, whereas netrin binds through multiple domains to both receptors. We examined the functional consequences of removing the netrin binding and nonbinding domains from Unc5 in vitro and in vivo. In human embryonic kidney 293 cells, removal of the netrin binding second Ig domain causes an increase in basal tyrosine phosphorylation, whereas removal of the netrin nonbinding thrombospondin domains decreases tyrosine phosphorylation. Moreover, experiments in Caenorhabditis elegans indicate that both netrin binding and nonbinding domains are necessary for phenotypic rescue of an unc-5 loss of function mutation.

Cloning, expression, and characterization of the Galalpha 1,3Gal high affinity lectin from the mushroom Marasmius oreades.

The purification and unique carbohydrate binding properties, including blood group B-specific agglutination and preferential binding to Galalpha1,3Gal-containing sugar epitopes, of the Marasmius oreades agglutinin (MOA) are reported in an accompanying paper (Winter, H. C., Mostafapour, K., and Goldstein, I. J. (2002) J. Biol. Chem. 277, 14996-15001). Here we describe the cloning, characterization, and expression of MOA. MOA was digested with trypsin and endoproteinase Asp-N, and the peptide fragments were purified by high performance liquid chromatography. Amino acid sequence data were obtained for eight peptides. Using oligonucleotides deduced from the peptide sequences for a reverse transcriptase-PCR, a 41-base pair cDNA was obtained. The 41-base pair fragment allowed the generation a full-length cDNA using 5' and 3' rapid amplification of cDNA ends. MOA cDNA encodes a protein of 293 amino acids that contains a ricin domain. These carbohydrate binding domains were first described in subunits of bacterial toxins and are also commonly found in polysaccharide-degrading enzymes. Whereas these proteins are known to display a variety of sugar binding specificities, none to date are known to share MOA's high affinity for Galalpha1,3Gal and Galalpha1,3Galbeta1,4GlcNAc. Recombinantly expressed and purified MOA retains the specificity and affinity observed with the native protein. This study provides the basis for analyzing the underlying cause for the unusual binding specificity of MOA.