IbpA DR2 subunit immunization protects calves against Histophilus somni pneumonia.

Histophilus somni is a prevalent cause of pneumonia and septicemia in cattle. Yet evidence for protection against pneumonia by current vaccines is controversial. We have identified a new H. somni virulence factor, IbpA. Previous studies implicated three likely protective subunits or domains in IbpA (A3, A5, and DR2), which were expressed as recombinant GST fusion proteins and purified for systemic vaccination of calves. After two subcutaneous immunizations, calves were challenged intrabronchially with virulent H. somni strain 2336 and clinical signs were monitored for four days before necropsy. Serum samples were collected throughout. At necropsy, the area of gross pneumonia was estimated, bronchial lavage fluid was collected, lesions were cultured and tissue samples were fixed for histopathology. Results showed that calves immunized with IbpA DR2 had a statistically lower percentage of lung with gross lesions than controls, fewer histologic abnormalities in affected areas and no H. somni isolated from residual pneumonic lesions. Calves immunized with the control GST vaccine, IbpA3 or IbpA5 had larger H. somni positive pneumonic lesions. ELISA results for serum antibodies showed that calves immunized with the IbpA DR2 antigen had high IgG1 and IgG2 and lowest IgE responses to the immunizing antigen. Specific IgG responses were also high in the bronchial lavage fluid. High specific serum IgE responses were previously shown to be associated with more severe pneumonia, but high IgG specific anti-IbpA DR2 responses seem to be critically related to protection. Since the IbpA DR2 Fic motif has been shown to cause bovine alveolar cells to retract, we tested the neutralizing ability of pooled serum from the IbpA DR2 immunized group. This pooled serum reduced cytotoxicity by 75-80%, suggesting that the protection was due to antibody neutralization of IbpA cytotoxicity, at least in part. Therefore, IbpA DR2 appears to be an important protective antigen of H. somni. The study shows, for the first time, that immunization with a purified Fic protein protects against disease in a natural host.

RNA interference of gene expression (RNAi) in cultured Drosophila cells.

RNA interference (RNAi) can be used to silence genes in a number of taxa, including plants, nematodes, protozoans, flies, and mammals represented by mouse embryos and cultured mammalian cells. To investigate signal transduction pathways, we used RNAi on Drosophila-cultured cells, which affords the opportunity to study protein function in a simple, well-defined cell culture system. Furthermore, the results obtained from experiments performed on cultured cells can be confirmed and extended in the whole organism, which, in the case of Drosophila, is also RNAi responsive. RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce posttranscriptional gene silencing in a highly specific manner. This silencing is efficacious and long-lived, as it is passed to subsequent generations in insect cell culture. To date, all Drosophila cell lines tested (S2, KC, BG2-C6, and Shi) respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested (more than 15) have been effective at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss-of-function studies applicable to a wide variety of questions.

The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton.

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.

The crystal structure of adenylosuccinate lyase from Pyrobaculum aerophilum reveals an intracellular protein with three disulfide bonds.

Adenylosuccinate lyase catalyzes two separate reactions in the de novo purine biosynthetic pathway. Through its dual action in this pathway, adenylosuccinate lyase plays an integral part in cellular replication and metabolism. Mutations in the human enzyme can result in severe neurological disorders, including mental retardation with autistic features. The crystal structure of adenylosuccinate lyase from the hyperthermophilic archaebacterium Pyrobaculum aerophilum has been determined to 2.1 A resolution. Although both the fold of the monomer and the architecture of the tetrameric assembly are similar to adenylosuccinate lyase from the thermophilic eubacterium Thermotoga maritima, the archaebacterial lyase contains unique features. Surprisingly, the structure of adenylosuccinate lyase from P. aerophilum reveals that this intracellular protein contains three disulfide bonds that contribute significantly to its stability against thermal and chemical denaturation. The observation of multiple disulfide bonds in the recombinant form of the enzyme suggests the need for further investigations into whether the intracellular environment of P. aerophilum, and possibly other hyperthermophiles, may be compatible with protein disulfide bond formation. In addition, the protein is shorter in P. aerophilum than it is in other organisms. This abbreviation results from an internal excision of a cluster of helices that may be involved in protein-protein interactions in other organisms and may relate to the observed clinical effects of human mutations in that region.

Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity.

A Drosophila homolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin superfamily member, was isolated by its affinity to Dock, an SH3/SH2 adaptor protein required for axon guidance. Dscam binds directly to both Dock's SH2 and SH3 domains. Genetic studies revealed that Dscam, Dock and Pak, a serine/threonine kinase, act together to direct pathfinding of Bolwig's nerve, containing a subclass of sensory axons, to an intermediate target in the embryo. Dscam also is required for the formation of axon pathways in the embryonic central nervous system. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conserved architecture containing variable Ig and transmembrane domains. Alternative splicing can potentially generate more than 38,000 Dscam isoforms. This molecular diversity may contribute to the specificity of neuronal connectivity.

Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways.

We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating "knock-out" phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1-4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project.

Positive versus negative signaling of LET-23: regulation through the adaptor protein, SEM-5.

Worby and Margolis highlight advances in our understanding of signaling from growth factor receptors using the worm Caenorhabditis elegans as a model organism. ARK-1, a cytoplasmic tyrosine kinase, appears to be a negative regulator of multiple pathways in C. elegans. The authors discuss several models for how this negative regulation may occur. The adaptor protein (Grb2 in mammals or SEM-5 in C. elegans) may serve as a regulated scaffold for the binding of other signaling proteins that include both positive (Ras) and negative (ACK) regulators. Thus, Grb2 may function in a cellular decision point for transducing the incoming signals.

His68 and His141 are critical contributors to the intersubunit catalytic site of adenylosuccinate lyase of Bacillus subtilis.

Mutant adenylosuccinate lyases of Bacillus subtilis were prepared by site-directed mutagenesis with replacements for His141, previously identified by affinity labeling as being in the active site [Lee, T. T., Worby, C., Dixon, J. E., and Colman, R. F. (1997) J. Biol. Chem. 272, 458-465]. Four substitutions (A, L, E, Q) yield mutant enzyme with no detectable catalytic activity, while the H141R mutant is about 10(-)5 as active as the wild-type enzyme. Kinetic studies show, for the H141R enzyme, a Km that is only 3 times that of the wild-type enzyme. Minimal activity was also observed for mutant enzymes with replacements for His68 [Lee, T. T., Worby, C., Bao, Z. -Q., Dixon, J. E., and Colman, R. F. (1998) Biochemistry 37, 8481-8489]. Measurement of the reversible binding of radioactive adenylosuccinate by inactive mutant enzymes with substitutions at either position 68 or 141 shows that their affinities for substrate are decreased by only 10-40-fold. These results suggest that His141, like His68, plays an important role in catalysis, but not in substrate binding. Evidence is consistent with the hypothesis that His141 and His68 function, respectively, as the catalytic base and acid. Circular dichroism spectroscopy and gel filtration chromatography conducted on wild-type and all His141 and His68 mutants reveal that none of the mutant enzymes exhibits major structural changes and that all the enzymes are tetramers. Mixing inactive His141 with inactive His68 mutant enzymes leads to striking increases in catalytic activity. This complementation of mutant enzymes indicates that His141 and His68 come from different subunits to form the active site. A tetrameric structure of adenylosuccinate lyase was constructed by homology modeling based on the known structures in the fumarase superfamily, including argininosuccinate lyase, delta-crystallin, fumarase, and aspartase. The model suggests that each active site is constituted by residues from three subunits, and that His141 and His68 come from two different subunits.

Evidence for regulated expression of neuropeptide Y gene by rat and human cultured astrocytes.

A series of studies from our laboratory have established that fetal rat and human neuropeptide Y (NPY) cortical neurons in aggregate cultures are differentially regulated. In a preliminary study we found that primary astrocytes produce substantial amounts of immunoreactive (IR) NPY. We addressed the question: Is astrocyte production of NPY-IR a regulated process? The effects of brain-derived neurotrophic factor (BDNF, 50 ng/ml), basic fibroblast growth factor (bFGF), substance P (1 microM), forskolin (10 microM), or phorbol 12-myristate-13-acetate (PMA, 20 nM) on NPY-IR production was tested on rat and human primary astrocyte cultures. Of these agents, PMA and bFGF markedly induced NPY-IR production by rat as well as human astrocytes, forskolin induced NPY-IR production by human but not rat astrocytes, and neither BDNF nor substance P induced NPY-IR production by rat or human astrocytes. The molecular size of PMA-induced NPY-IR was found to be consistent with that of proNPY. Moreover, PMA induced the accumulation of mRNA corresponding in size to the neuronal NPY-mRNA. Immunocytochemical analysis of human post-mortem neocortex revealed co-existence of NPY-IR with astrocyte markers. These results indicate that cultured astrocytes express NPY gene in a regulated manner and they support our proposition that in situ reactive astrocytes may express NPY gene under some physiological/pathological conditions.

Implication of His68 in the substrate site of Bacillus subtilis adenylosuccinate lyase by mutagenesis and affinity labeling with 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate.

Adenylosuccinate lyase of Bacillus subtilis is inactivated by 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate (2-BDB-TAMP) at pH 7.0. As the reagent concentration is increased, a maximum rate constant is approached, indicative of reversible enzyme-reagent complex formation (KR = 68 +/- 9 microM) prior to irreversible modification (kmax = 0.081 +/- 0.004 min-1). Complete inactivation occurs concomitant with about 1 mol of 2-BDB-[14C]TAMP incorporated/mol of enzyme subunit. Adenylosuccinate, or a combination of AMP and fumarate, decreases the inactivation rate and reduces incorporation of [14C] reagent, whereas either AMP or fumarate alone is much less effective. These observations suggest that 2-BDB-TAMP attacks the adenylosuccinate binding site. Proteolytic digestion of inactivated enzyme, followed by purification of the digest by HPLC, yields the radioactive peptide Ile62-Ala72, in which Arg67 and His68 are the most likely targets. Thus 2-BDB-TAMP reacts with adenylosuccinate lyase at a site distinct from the His141 attacked by 6-BDB-TAMP (Lee, Worby, Dixon, and Colman (1997) J. Biol. Chem. 272, 458-465). Site-directed mutagenesis was used to construct mutant enzymes with replacements for both Arg67 and His68, and either Arg67 or His68. The R67M mutant enzyme has almost the same specific activity as the wild-type enzyme under standard assay conditions, whereas the single mutant H68Q and double mutant R67M-H68Q enzymes exhibit specific activities that are decreased more than 100-fold. These results indicate that while Arg67 and His68 may both be in the region of the substrate site, only His68 is important for the catalytic activity of B. subtilis adenylosuccinate lyase. A role is proposed for His68 as a general acid-base catalyst.

Activated transcription of the human neuropeptide Y gene in differentiating SH-SY5Y neuroblastoma cells is dependent on transcription factors AP-1, AP-2alpha, and NGFI.

Activated transcription of the human neuropeptide Y gene (NPY) was investigated in SH-SY5Y neuroblastoma cells at the onset of sympathetic neuronal differentiation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) and serum or by nerve growth factor (NGF). As determined by transient expression, two NGF response elements (REs) were required for transcription induced by NGF in SH-SY5Y cells with stable expression of an exogenous NGF receptor TRK-A gene (SH-SY5Y/trk). TPA treatment in the presence of serum induced NPY transcription in both wild-type SH-SY5Y (SH-SY5Y/wt) and SH-SY5Y/trk cells. A TPA RE (TRE), overlapping the proximal NGF RE, was identified by expression of the v-Jun oncoprotein that enhanced NPY transcription. Suppression of TPA-induced NPY transcription was obtained by expression of a dominant negative Jun protein, selective protein kinase C inhibition, or introduction of a mutated TRE, whereas NGF-induced NPY transcription was inhibited to a lesser degree. The transcription factor AP-2alpha was shown to bind cooperatively to the NPY promoter with either AP-1 or NGFI-A to the shared TRE and NGF RE and to the distal NGF RE, respectively. These results show that transcription factors AP-1, AP-2alpha, and NGFI-A are involved in activated NPY transcription during the onset of neuronal differentiation.

Identification and characterization of GFRalpha-3, a novel Co-receptor belonging to the glial cell line-derived neurotrophic receptor family.

A new family of neuronal survival factors comprised of glial cell line-derived neurotrophic factor (GDNF) and neurturin has recently been described (Kotzbauer, P. T., Lampe, P. A., Heuckeroth, R. O., Golden, J. P., Creedon, D. J., Johnson, E. M., Jr., and Milbrandt, J. (1997) Nature 384, 467-470). These molecules, which are related to transforming growth factor-beta, are important in embryogenesis and in the survival of distinct neuronal populations. These molecules signal through a novel receptor system that includes the Ret receptor tyrosine kinase, a ligand (i.e. GDNF or neurturin), and an accessory glycosyl-phosphatidylinositol-linked molecule that is responsible for high affinity binding of the ligand. Two accessory molecules denoted GDNF family receptor 1 and 2 (GFRalpha-1 and GFRalpha-2) have been described that function in GDNF and neurturin signaling complexes. We have identified a novel co-receptor belonging to this family based on similarity to GFRalpha-1, which we have named GFRalpha-3. GFRalpha-3 displays 33% amino acid identity with GFRalpha-1 and 36% identity with GFRalpha-2. Despite the similarity of GFRalpha-3 to GFRalpha-1 and GFRalpha-2, it is unable to activate Ret in conjunction with GDNF, suggesting that there are likely additional undiscovered ligands and/or Ret-like receptors to be identified. GFRalpha-3 is anchored to the cell membrane by a phosphatidylinositol-specific phospholipase C-resistant glycosyl-phosphatidylinositol linkage. GFRalpha-3 is highly expressed by embryonic day 11 but is not appreciably expressed in the adult mouse. In situ hybridization analyses demonstrate that GFRalpha-3 is located in dorsal root ganglia and the superior cervical sympathetic ganglion. Comparison of the expression patterns of GFRalpha-3 and Ret suggests that these molecules could form a receptor pair and interact with GDNF family members to play unique roles in development.

Identification of His141 in the active site of Bacillus subtilis adenylosuccinate lyase by affinity labeling with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate.

Adenylosuccinate lyase of Bacillus subtilis is inactivated by 25-400 microM 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial inactivation rate constant exhibits nonlinear dependence on the concentration of 6-BDB-TAMP, implying there is reversible formation of enzyme-reagent complex (K(I) = 30 +/- 4 microM) prior to irreversible modification (kmax = 0.139 +/- 0.005 min(-1)). The tetrameric enzyme incorporates about 1 mol of 6-BDB-[32P]TAMP per mol of enzyme subunit concomitant with complete inactivation. Protection against inactivation and incorporation of [32P]reagent is provided by adenylosuccinate or a combination of AMP and fumarate, whereas either AMP or fumarate alone is much less effective. These observations suggest that 6-BDB-TAMP targets the adenylosuccinate-binding site. Hydrolyzed 6-BDB-TAMP is a competitive inhibitor with respect to adenylosuccinate in the catalytic reaction and also decreases the rate of inactivation by 6-BDB-TAMP. These results account for the decrease in the inactivation rate as the reaction of 6-BDB-TAMP with the enzyme proceeds. Purification by chromatography on dihydroxyboryl-agarose and high performance liquid chromatography of the tryptic digest of inactivated enzyme yields a single radioactive peptide, Thr140-Phe150, as determined by gas-phase sequencing. Modified His141 is the reaction product of 6-BDB-TAMP and adenylosuccinate lyase. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analog in modifying His141 in the substrate-binding site of adenylosuccinate lyase, where it may serve as a general base accepting a proton from the succinyl group during catalysis.

Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis.

The receptor tyrosine kinase RET functions during the development of the kidney and the enteric nervous system, yet no ligand has been identified to date. This report demonstrates that the glial cell line-derived neurotrophic factor (GDNF) activates RET, as measured by tyrosine phosphorylation of the intracellular catalytic domain. GDNF also binds RET with a dissociation constant of 8 nM, and 125I-labeled GDNF can be coimmunoprecipitated with anti-RET antibodies. In addition, exogenous GDNF stimulates both branching and proliferation of embryonic kidneys in organ culture, whereas neutralizing antibodies against GDNF inhibit branching morphogenesis. These data indicate that RET and GDNF are components of a common signaling pathway and point to a role for GDNF in kidney development.

Glial cell line-derived neurotrophic factor signals through the RET receptor and activates mitogen-activated protein kinase.

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-beta family of growth factors, was first identified by its ability to promote the survival of midbrain dopaminergic neurons in culture. We demonstrate that GDNF treatment of several neuroblastoma cell lines leads to dose-dependent tyrosine phosphorylation of the RET receptor and that other transforming growth factor-beta family members are not able to activate the RET receptor. GDNF treatment of neuroblastoma cells also results in increased transcription of an Elk luciferase reporter gene, suggesting that GDNF activates the mitogen-activated protein kinase signal transduction pathway.

Oncogenic RET receptors display different autophosphorylation sites and substrate binding specificities.

The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in neural crest as well as kidney development. Genetic studies have demonstrated that germ line mutations in the ret oncogene are the direct cause of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC), and Hirschsprung's disease. However, despite the large body of genetic and biological evidence suggesting the importance of RET in development and neoplastic processes, the signal transduction mechanisms of RET remain unknown. To begin to understand the molecular mechanisms of the disease states caused by mutations in RET, the patterns of autophosphorylation of the wild-type RET and the MEN mutants were studied using site-directed mutagenesis and phosphopeptide mapping. Among the 6 autophosphorylation sites found in the wild-type RET receptor, the MEN2B mutant lacked phosphorylation at Tyr-1096, leading to decreased Grb2 binding, while simultaneously creating a new phosphorylation site. These changes in autophosphorylation suggest that the MEN2B mutation may result in the more aggressive MEN2B phenotype by altering the receptor's signaling capabilities.

Transcriptional vs. posttranscriptional control of neuropeptide Y gene expression.

Human neuropeptide Y (NPY) gene expression occurs exclusively in the central and peripheral nervous systems requiring complex cell-specific regulation. In this study we have examined the effect of modulating the second messenger systems involving protein kinase A and protein kinase C on the expression of the NPY gene in different neuronal cell types. We report that the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and forskolin on a neuroblastoma cell line (LA-N-5) and a pheochromocytoma cell line (PC12) are mediated through both increased transcription of the NPY gene and through stabilization of NPY messenger RNA (mRNA). After 8 h of treatment TPA and forskolin increase the steady-state level of NPY mRNA 10- and 12-fold in LA-N-5 and PC12 cells, respectively. This response in neuroblastoma cells is due to an increase in the half-life of NPY mRNA. The response in PC12 cells is mediated by both increased mRNA stability and increased transcription. Transient transfection analyses using PC12 cells indicate that only 51 base pairs 5' to the transcription start site are necessary for the TPA and forskolin induced transcriptional response. Thus, these experiments demonstrate that TPA and forskolin effect the regulation of the NPY gene via transcriptional and posttranscriptional mechanisms in a cell-specific manner.

Transcriptional regulation of the human neuropeptide Y gene by nerve growth factor.

Neuropeptide Y (NPY) is the most ubiquitously expressed peptide in the mammalian nervous system. Transcription of the NPY gene in PC12 cells is regulated by a number of agents, including the neurotrophic peptide nerve growth factor (NGF). In this paper, we define the cis-acting promoter elements which respond to NGF and characterize the trans-acting factors which interact with these sequences. The NGF-responsive elements of the NPY gene lie between nucleotides -87 and -36. At least four proteins interact with this promoter region. One of these proteins interacts with a CT-rich sequence centered at position -51, which closely abuts a binding site for transcription factor AP-2 centered at position -63. Two newly characterized factors bind between positions -87 and -58. These proteins are expressed in a tissue-specific manner and, together with the other binding activities, modulate the transcriptional activity of the NPY gene. These results suggest that the concerted interplay of these proteins, in response to NGF, increases the transcriptional activity of the NPY gene.