Ler of pathogenic Escherichia coli forms toroidal protein-DNA complexes.

Enteropathogenic and enterohaemorrhagic Escherichia coli are related pathotypes of bacteria that cause acute watery diarrhoea and haemorrhagic colitis, respectively, and enterohaemorrhagic E. coli can lead to a serious complication known as haemolytic uraemic syndrome. In both bacteria the global regulatory protein Ler controls virulence. The ler gene is found within the locus of enterocyte effacement, or LEE, encoding a type III secretion system necessary for injecting effector proteins into intestinal epithelial cells and causing net secretory diarrhoea. The nucleoid-associated protein H-NS silences, whereas Ler serves as an anti-silencer of, multiple LEE operons. Although Ler has a higher affinity for DNA than does H-NS, the precise molecular mechanism by which Ler increases LEE transcription remains to be determined. In this report we investigate the oligomerization activity of Ler. In solution, Ler forms dimers and soluble aggregates of up to 5000 kDa molecular mass, and appears to oligomerize more readily than the related protein H-NS. An insertional mutation into the Ler linker region diminished oligomerization activity. Despite being proteins of similar mass and having homologous DNA-binding domains, Ler and H-NS complexed to DNA migrated to distinct locations, as determined by an electrophoretic mobility shift assay, implying that the related proteins form different 3D shapes in the presence of DNA. Lastly, we present electron microscopy images of toroidal Ler-DNA structures that are predicted to be involved in stimulating gene expression.

Context Specificity in Causal Signaling Networks Revealed by Phosphoprotein Profiling.

Signaling networks downstream of receptor tyrosine kinases are among the most extensively studied biological networks, but new approaches are needed to elucidate causal relationships between network components and understand how such relationships are influenced by biological context and disease. Here, we investigate the context specificity of signaling networks within a causal conceptual framework using reverse-phase protein array time-course assays and network analysis approaches. We focus on a well-defined set of signaling proteins profiled under inhibition with five kinase inhibitors in 32 contexts: four breast cancer cell lines (MCF7, UACC812, BT20, and BT549) under eight stimulus conditions. The data, spanning multiple pathways and comprising ∼70,000 phosphoprotein and ∼260,000 protein measurements, provide a wealth of testable, context-specific hypotheses, several of which we experimentally validate. Furthermore, the data provide a unique resource for computational methods development, permitting empirical assessment of causal network learning in a complex, mammalian setting.

Physiological roles of glutathione s-transferases in soybean root nodules.

Glutathione S-transferases (GSTs) are ubiquitous enzymes that catalyze the conjugation of toxic xenobiotics and oxidatively produced compounds to reduced glutathione, which facilitates their metabolism, sequestration, or removal. We report here that soybean (Glycine max) root nodules contain at least 14 forms of GST, with GST9 being most prevalent, as measured by both real-time reverse transcription-polymerase chain reaction and identification of peptides in glutathione-affinity purified extracts. GST8 was prevalent in stems and uninfected roots, whereas GST2/10 prevailed in leaves. Purified, recombinant GSTs were shown to have wide-ranging kinetic properties, suggesting that the suite of GSTs could provide physiological flexibility to deal with numerous stresses. Levels of GST9 increased with aging, suggesting a role related to senescence. RNA interference studies of nodules on composite plants showed that a down-regulation of GST9 led to a decrease in nitrogenase (acetylene reduction) activity and an increase in oxidatively damaged proteins. These findings indicate that GSTs are abundant in nodules and likely function to provide antioxidant defenses that are critical to support nitrogen fixation.