Isolation and characterization of a human cDNA for mRNA 5'-capping enzyme.

The human mRNA 5'-capping enzyme cDNA was identified. Three highly related cDNAs, HCE1 (human mRNAcappingenzyme1), HCE1A and HCE1B , were isolated from a HeLa cDNA library. The HCE1 cDNA has the longest ORF, which can encode a 69 kDa protein. A short region of 69 bp in the 3'-half of the HCE1 ORF was missing in HCE1A and HCE1B , and, additionally, HCE1B has an early translation termination signal, which suggests that the latter two cDNAs represent alternatively spliced product. When expressed in Escherichia coli as a fusion protein with glutathione S -transferase, Hce1p displayed both mRNA 5'-triphosphatase (TPase) and mRNA 5'-guanylyltransferase (GTase) activities, and it formed a cap structure at the 5'-triphosphate end of RNA, demonstrating that it indeed specifies an active mRNA 5'-capping enzyme. The recombinant proteins derived from HCE1A and HCE1B possessed only TPase activity. When expressed from ADH1 promoter, HCE1 but not HCE1A and HCE1B complemented Saccharomyces cerevisiae CEG1 and CET1 , the genes for GTase and TPase, respectively. These results demonstrate that the N-terminal part of Hce1p is responsible for TPase activity and the C-terminal part is essential for GTase activity. In addition, the human TPase domain cannot functionally substitute for the yeast enzyme in vivo.     

The Caenorhabditis elegans mRNA 5'-capping enzyme. In vitro and in vivo characterization

Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference in C. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.     

Divergent regulation of gonadotropin subunit mRNA levels by androgens in the female rat.

To examine the effects of gonadal steroids on the pretranslational regulation of the gonadotropin subunits in the female, adult female rats, beginning 7 or 28 days after ovariectomy, received daily injections of testosterone propionate (T), dihydrotestosterone propionate (D), or estradiol benzoate (E) for 7 days. Intact cycling females and ovariectomized rats that received vehicle served as controls. Serum was obtained for LH and FSH levels to assess changes in gonadotropin secretion. Total RNA from individual rats was recovered and analyzed by blot hybridization with specific radiolabeled cDNA probes for the alpha, LH beta, and FSH beta subunits. Autoradiographic bands were quantitated and standardized to mRNA levels in the intact animals. Ovariectomy resulted in a rise in serum gonadotropin levels and all three gonadotropin subunit mRNA levels. Estrogen replacement resulted in suppression of alpha, LH beta, and FSH beta mRNAs whether given at 7 or 28 days after ovariectomy. In contrast, whereas androgen replacement decreased alpha and LH beta mRNAs, D or T did not consistently suppress FSH beta mRNAs. We conclude that chronic estrogen administration to the castrated female rat uniformly suppresses all three gonadotropin subunit mRNA levels. In female rats, as in male rats, chronic androgen administration fails to negatively regulate FSH beta mRNAs.     

G protein beta 5 subunit interactions with alpha subunits and effectors

When the beta(5) (short form) and gamma(2) subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged alpha(i) in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged alpha(i) and resulted in purification of beta(5)gamma(2) to near homogeneity. Subsequent anion-exchange chromatography of beta(5)gamma(2) resulted in resolution of beta(5) from gamma(2) and further purification of beta(5). The purified beta(5) eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of gamma. beta(5) monomer could be assembled with partially purified hexahistidine-tagged gamma(2) in vitro to form a functional dimer that could selectively activate PLC beta2 but not PLC beta3. alpha(o)-GDP inhibited activation of PLC beta2 by beta(5)gamma(2) supporting the idea that beta(5)gamma(2) can bind to alpha(o). beta(5) monomer and beta(5)gamma(2) only supported a small degree of ADP ribosylation of alpha(i) by pertussis toxin (PTX), but beta(5) monomer was able to compete for beta(1)gamma(2) binding to alpha(i) and alpha(o) to inhibit PTX-catalyzed ADP ribosylation. These data indicate that beta(5) functionally interacts with PTX-sensitive GDP alpha subunits and that beta(5) subunits can be assembled with gamma subunits in vitro to reconstitute activity and also support the idea that there are determinants on beta subunits that are selective for even very closely related effectors.     

Multicolor BiFC analysis of competition among G protein beta and gamma subunit interactions

We have applied multicolor BiFC to study the association preferences of G protein beta and gamma subunits in living cells. Cells co-express multiple isoforms of beta and gamma subunits, most of which can form complexes. Although many betagamma complexes exhibit similar properties when assayed in reconstituted systems, knockout experiments in vivo suggest that individual isoforms have unique functions. BiFC makes it possible to correlate betagamma complex formation with functionality in intact cells by comparing the amounts of fluorescent betagamma complexes with their abilities to modulate effector proteins. The relative predominance of specific betagamma complexes in vivo is not known. To address this issue, multicolor BiFC can determine the association preferences of beta and gamma subunits by simultaneously visualizing the two fluorescent complexes formed when beta or gamma subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete to interact with limiting amounts of a common gamma or beta subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). Multicolor BiFC also makes it possible to determine the roles of interacting proteins in the subcellular targeting of complexes, study the formation of protein complexes that are unstable under isolation conditions, determine the roles of co-expressed proteins in regulating the association preferences of interacting proteins, and visualize dynamic events affecting multiple protein complexes. These approaches can be applied to studying the assembly and functions of a wide variety of protein complexes in the context of a living cell.     

The plasticity of fungal interactions

Their hyphal structure, the common events of hybridization and horizontal gene transfer, as well as intimate associations with prokaryotes (including endobiotic bacteria) and cooperation with eukaryotes have made fungi very flexible at the genetic, physiological, and ecological levels. It is manifested with the fungal ability to perfectly exploit existing nutrient sources and plastically fit into a changing environment. Although the links between fungi and other ecosystem components are rarely clearly visible and unambiguous, fungi can be ecosystem buffers playing a homeostatic role throughout global ecosystems, reacting to changes in various ways, not only by modifications of gene expression but also by nuclear status and “extended phenotype”. The goal of this review is to underline some ecological interactions involving fungi and other organisms and to indicate high fungal plasticity in terms of ontogenetic perspective.     

Letter: Troponin subunit interactions

Subunit interactions of the troponin complex are displayed by the in vitro aggregates formed by tropomyosin and the components of troponin. Certain linkages are specifically affected by calcium. These findings are consistent with a simple steric scheme for the functioning of the complex.     

Structural interactions between transmembrane helices 4 and 5 of subunit a and the subunit c ring of Escherichia coli ATP synthase

Subunit a plays a key role in promoting H+ transport and the coupled rotary motion of the subunit c ring in F1F0-ATP synthase. H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F0 subunit c. H+ are thought to reach Asp-61 via aqueous pathways mapping to the surfaces of TMHs 2-5 of subunit a. TMH4 of subunit a is thought to pack close to TMH2 of subunit c based upon disulfide cross-link formation between Cys substitutions in both TMHs. Here we substituted Cys into the fifth TMH of subunit a and the second TMH of subunit c and tested for cross-linking using bis-methanethiosulfonate (bis-MTS) reagents. A total of 62 Cys pairs were tested and 12 positive cross-links were identified with variable alkyl length linkers. Cross-linking was achieved near the middle of the bilayer for the Cys pairs a248C/c62C, a248C/ c63C, a248C/c65C, a251C/c57C, a251C/c59C, a251C/c62C, a252C/c62C, and a252C/c65C. Cross-linking was achieved near the cytoplasmic side of the bilayer for Cys pairs a262C/c53C, a262C/c54C, a262C/c55C, and a263C/c54C. We conclude that both aTMH4 and aTMH5 pack proximately to cTMH2 of the c-ring. In other experiments we demonstrate that aTMH4 and aTMH5 can be simultaneously cross-linked to different subunit c monomers in the c-ring. Five mutants showed pH-dependent cross-linking consistent with aTMH5 changing conformation at lower pH values to facilitate cross-linking. We suggest that the pH-dependent conformational change may be related to the proposed role of aTMH5 in gating H+ access from the periplasm to the cAsp-61 residue in cTMH2.     

Regulation of fungal gene expression via short open reading frames in the mRNA 5'untranslated region

We review how the expression of fungal mRNAs can be controlled by ribosome interactions with short upstream open reading frames (uORFs) within the 5'untranslated region. The efficiency of uAUG recognition modulates the impact of a uORF but steps during and after translation of the uORF also influence uORF function. The post-termination behaviour of ribosomes, therefore, plays a major role in determining the expression level of these main ORFs. Translation of a uORF can produce a cis-acting peptide that causes effector molecule-dependent stalling of the ribosomes at the end of the uORF. In other cases it is the length or position, or other features of the uORF, rather than the peptide it encodes, that determine the efficiency with which ribosomes reinitiate translation downstream of it. Whether the form of the ribosome that resumes scanning after termination is the 40S subunit alone or the entire 80S ribosome is not known. Translation of the uORF can also control gene expression by affecting the stability of the mRNA. Finally, trans-acting factors may participate in the regulatory mechanisms. Future work will need not only to provide more information on the mechanisms underlying the known cases of uORF-mediated control but also to define the full complement of uORF-containing mRNAs in at least one fungal organism.     

Balancing multiple mutualists: asymmetric interactions among plants, arbuscular mycorrhizal fungi, and fungal endophytes

Most organisms engage in beneficial interactions with other species; however, little is known regarding how individuals balance the competing demands of multiple mutualisms. Here we examine three-way interactions among a widespread grass, Schedonorus phoenix , a protective fungal endophyte aboveground, Neotyphodium coenophialum , and nutritional symbionts (arbuscular mycorrhizal fungi) belowground. In a greenhouse experiment, we manipulated the presence/absence of both fungi and applied a fertilizer treatment to individual plants. Endophyte presence in host plants strongly reduced mycorrhizal colonization of roots. Additionally, for plants with the endophyte, the density of endophyte hyphae was negatively correlated with mycorrhizal colonization, suggesting a novel role for endophyte abundance in the interaction between the symbionts. Endophyte presence increased plant biomass, and there was a positive correlation between endophyte hyphal density and plant biomass. The effects of mutualists were asymmetric: mycorrhizal fungi treatments had no significant impact on the endophyte and negligible effects on plant biomass. Fertilization affected all three species – increasing plant biomass and endophyte density, but diminishing mycorrhizal colonization. Mechanisms driving negative effects of endophytes on mycorrhizae may include inhibition via endophyte alkaloids, altered nutritional requirements of the host plant, and/or temporal and spatial priority effects in the interactions among plants and multiple symbionts.     

Cytology of fungal pathogens and plant-host interactions

Imaging plays a unique role in fungal cell biology and phytopathology by allowing for the documentation of molecular structure in individual fixed and living cells. Advances in fluorescence laser techniques, including confocal and multiphoton microscopy, are opening new avenues for cellular exploration. These techniques hold tremendous potential for studies of host-pathogen interactions including the use of genetically encoded markers such as green fluorescent protein, in situ hybridization and fluorescence resonance energy transfer.     

Divergent Inhibitor Susceptibility among Airway Lumen-Accessible Tryptic Proteases

Tryptic serine proteases of bronchial epithelium regulate ion flux, barrier integrity, and allergic inflammation. Inhibition of some of these proteases is a strategy to improve mucociliary function in cystic fibrosis and asthmatic inflammation. Several inhibitors have been tested in pre-clinical animal models and humans. We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects. To establish relative potencies and modes of action, we compared inactivation of human prostasin, matriptase, airway trypsin-like protease (HAT), and β-tryptase by nafamostat, camostat, bis(5-amidino-2-benzimidazolyl)methane (BABIM), aprotinin, and benzamidine. Nafamostat achieved complete, nearly stoichiometric and very slowly reversible inhibition of matriptase and tryptase, but inhibited prostasin less potently and was weakest versus HAT. The IC₅₀ of nafamostat’s leaving group, 6-amidino-2-naphthol, was >10⁴-fold higher than that of nafamostat itself, consistent with suicide rather than product inhibition as mechanisms of prolonged inactivation. Stoichiometric release of 6-amidino-2-naphthol allowed highly sensitive fluorometric estimation of active-site concentration in preparations of matriptase and tryptase. Camostat inactivated all enzymes but was less potent overall and weakest towards matriptase, which, however was strongly inhibited by BABIM. Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase. Benzamidine was universally weak. Thus, each inhibitor profile was distinct. Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.     

Extracellular enzyme localization during interspecific fungal interactions

Abstract Confronted colonies of Phlebia radiata, P. rufa, Coriolus versicolor, Stereum hirsutum, Phanerochaete velutina and Hypholoma fasciculare showed spatially and temporally heterogeneous patterns of loccase-α-naphthol and peroxidase activities. These activities were coincident in axenic cultures. but were not always so during interaction. Confrontation between species resulted in induction of phenoloxidase activities, even within coenocytic colony regions of Phlebia species which were normally void of such activities in axenic culture. These events resulted in restriction of C. versicolor growth during interaction with P. rufa.