The Doc toxin and Phd antidote proteins of the bacteriophage P1 plasmid addiction system form a heterotrimeric complex.

The toxin (Doc) and antidote (Phd) proteins of the plasmid addiction system of bacteriophage P1 were purified as a complex. Cocrystals of the complex contained a 2:1 molar ratio of Phd:Doc as assayed by dye binding following SDS-polyacrylamide gel electrophoresis and as determined by amino acid analysis. Gel filtration and analytical ultracentrifugation revealed that the two addiction proteins interact in solution to form a P2D trimer composed of one Doc and two Phd molecules. These results support a model in which Phd inhibits the toxic activity of Doc by direct binding. Circular dichroism experiments showed that changes in secondary structure accompany formation of the heterotrimeric complex, raising the possibility that Phd may act by an allosteric mechanism. Studies of Phd and Doc molecules labeled with fluorescent energy donor and acceptor groups gave an equilibrium dissociation constant of about 0.8 microM(2) and a very short, sub second half-life of complex dissociation. As a consequence, low concentrations of free Doc toxin are likely to be present both transiently and in the steady state in cells containing the Phd antidote, making mechanisms of single-hit Doc toxicity improbable.     

c-fos Expression in the forebrain and brainstem of White Leghorn hens following osmotic and cardiovascular challenges

In chickens, hyperosmolality and hemorrhage increase hypothalamic vasotocin (AVT) gene expression and stimulate the secretion of AVT from the posterior pituitary gland. In this study, c-fos expression was used to identify areas in the forebrain and brainstem of the domestic chicken that are activated following acute osmotic stress and hemorrhage-induced hypotension. Conscious hens were osmotically stimulated by administering a single intraperitoneal injection of 3 M NaCl (5 ml/kg). Urethane-anesthetized hens were bled to a mean systemic arterial pressure of 80-90 mm Hg and maintained at this blood pressure for 1 h with additional bleedings as required. In both studies, the expression of c-fos was determined in control and experimental birds by using Northern blot analysis and in situ hybridization analysis. Osmotic stress and hemorrhage-induced hypotension increased c-fos expression in the same brain regions. Prominent structures in the forebrain that expressed c-fos mRNA following acute osmotic stress and hemorrhage-induced hypotension included the supraoptic nucleus and paraventricular nucleus and nuclei within the hypothalamus that are anterior and ventral to the third ventricle. In the chicken, this region includes the organum subseptale, the o. vasculosum laminae terminalis, and the nucleus septalis medialis. In the brainstem, following either injection of 3 M NaCl or hemorrhage-induced hypotension, increased c-fos expression was observed in the nucleus of the solitary tract, parabrachial nucleus, area postrema, and locus ceruleus. Thus, the chicken central nervous system appears to use shared neuronal circuitry to stimulate hypothalamic AVT release in response to disturbances in body fluid composition and decreases in either systemic blood pressure or volume.     

Self-Assembly of Phenylalanine Oligopeptides: Insights from Experiments and Simulations

Studies of peptide-based nanostructures provide general insights into biomolecular self-assembly and can lead material engineering toward technological applications. The diphenylalanine peptide (FF) self-assembles into discrete, hollow, well ordered nanotubes, and its derivatives form nanoassemblies of various morphologies. Here we demonstrate for the first time, to our knowledge, the formation of planar nanostructures with β-sheet content by the triphenylalanine peptide (FFF). We characterize these structures using various microscopy and spectroscopy techniques. We also obtain insights into the interactions and structural properties of the FF and FFF nanostructures by 0.4-μs, implicit-solvent, replica-exchange, molecular-dynamics simulations of aqueous FF and FFF solutions. In the simulations the peptides form aggregates, which often contain open or ring-like peptide networks, as well as elementary and network-containing structures with β-sheet characteristics. The networks are stabilized by polar and nonpolar interactions, and by the surrounding aggregate. In particular, the charged termini of neighbor peptides are involved in hydrogen-bonding interactions and their aromatic side chains form “T-shaped” contacts, as in three-dimensional FF crystals. These interactions may assist the FF and FFF self-assembly at the early stage, and may also stabilize the mature nanostructures. The FFF peptides have higher network propensities and increased aggregate stabilities with respect to FF, which can be interpreted energetically.     

New tryptophanase inhibitors: towards prevention of bacterial biofilm formation

Tryptophanase (tryptophan indole-lyase, Tnase, EC 4.1.99.1), a bacterial enzyme with no counterpart in eukaryotic cells, produces from L-tryptophan pyruvate, ammonia and indole. It was recently suggested that indole signaling plays an important role in the stable maintenance of multicopy plasmids. In addition, Tnase was shown to be capable of binding Rcd, a short RNA molecule involved in resolution of plasmid multimers. Binding of Rcd increases the affinity of Tnase for tryptophan, and it was proposed that indole is involved in bacteria multiplication and biofilm formation. Biofilm-associated bacteria may cause serious infections, and biofilm contamination of equipment and food, may result in expensive consequences. Thus, optimal and specific factors that interact with Tnase can be used as a tool to study the role of this multifunctional enzyme as well as antibacterial agents that may affect biofilm formation. Most known quasi-substrates inhibit Tnase at the mM range. In the present work, the mode of Tnase inhibition by the following compounds and the corresponding Ki values were: S-phenylbenzoquinone-L-tryptophan, uncompetitively, 101 microM; alpha-amino-2-(9,10-anthraquinone)-propanoic acid, noncompetitively, 174 microM; L-tryptophane-ethylester, competitively, 52 microM; N-acetyl-L-tryptophan, noncompetitively, 48 microM. S-phenylbenzoquinone-L-tryptophan and alpha-amino-2-(9,10-anthraquinone)-propanoic acid were newly synthesized.     

The E3 Ubiquitin-Ligase Bmi1/Ring1A Controls the Proteasomal Degradation of Top2α Cleavage Complex – A Potentially New Drug Target

Background

The topoisomerases Top1, Top2α and Top2β are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2β are subject to proteasomal degradation, this phenomena was not demonstrated for Top2α.

Methodology/Principal Findings

We show here that Top2α is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2β. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2α degradation, increases the persistence of Top2α-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2α in-vitro and cellular overexpression of Bmi1 increases drug induced Top2α ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2α ubiquitination and drug-induced Top2α degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner.

Conclusions/Significance

The discovery that poisoned Top2α is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1.     

Novel α‐ketoglutarate dioxygenase tfdA‐related genes are found in soil DNA after exposure to phenoxyalkanoic herbicides

Phenoxyalkanoic herbicides such as 2,4‐dichlorophenoxyacetate (2,4‐D), 2,4‐dichlorophenoxybutyrate (2,4‐DB) or mecoprop are widely used to control broad‐leaf weeds. Several bacteria have been reported to degrade these herbicides using the α‐ketoglutarate‐dependent, 2,4‐dichlorophenoxyacetate dioxygenase encoded by the tfdA gene, as the enzyme catalysing the first step in the catabolic pathway. The effects of exposure to different phenoxyalkanoic herbicides in the soil bacterial community and in the tfdA genes diversity were assessed using an agricultural soil exposed to these anthropogenic compounds. Total community bacterial DNA was analysed by terminal restriction fragment length polymorphism of the 16S rRNA and the tfdA gene markers, and detection and cloning of tfdA gene related sequences, using PCR primer pairs. After up to 4 months of herbicide exposure, significant changes in the bacterial community structure were detected in soil microcosms treated with mecoprop, 2,4‐DB and a mixture of both plus 2,4‐D. An impressive variety of novel tfdA gene related sequences were found in these soil microcosms, which cluster in new tfdA gene related sequence groups, unequally abundant depending on the specific herbicide used in soil treatment. Structural analysis of the putative protein products showed small but significant amino acid differences. These tfdA gene sequence variants are, probably, required for degradation of natural substrate(s) structurally related to these herbicides and their presence explains self‐remediation of soils exposed to phenoxyalkanoic herbicides.     

Hsp90 recognizes a common surface on client kinases

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.