Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the K_i in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.     

Quantitative phosphoproteomics of early elicitor signaling in Arabidopsis

Perception of general elicitors by plant cells initiates signal transduction cascades that are regulated by protein phosphorylation. The earliest signaling events occur within minutes and include ion fluxes across the plasma membrane, activation of MAPKs, and the formation of reactive oxygen species. The phosphorylation events that regulate these signaling cascades are largely unknown. Here we present a mass spectrometry-based quantitative phosphoproteomics approach that identified differentially phosphorylated sites in signaling and response proteins from Arabidopsis cells treated with either flg22 or xylanase. Our approach was sensitive enough to quantitate phosphorylation on low abundance signaling proteins such as calcium-dependent protein kinases and receptor-like kinase family members. With this approach we identified one or more differentially phosphorylated sites in 76 membrane-associated proteins including a number of defense-related proteins. Our data on phosphorylation indicate a high degree of complexity at the level of post-translational modification as exemplified by the complex modification patterns of respiratory burst oxidase protein D. Furthermore the data also suggest that protein translocation and vesicle traffic are important aspects of early signaling and defense in response to general elicitors. Our study presents the largest quantitative Arabidopsis phosphoproteomics data set to date and provides a new resource that can be used to gain novel insight into plant defense signal transduction and early defense response.     

Contrasting interactions between ethylene and abscisic acid in Rumex species differing in submergence tolerance

Complete submergence of flooding-tolerant Rumex palustris plants strongly stimulates petiole elongation. This escape response is initiated by the accumulation of ethylene inside the submerged tissue. In contrast, petioles of flooding-intolerant Rumex acetosa do not increase their elongation rate under water even though ethylene also accumulates when they are submerged. Abscisic acid (ABA) was found to be a negative regulator of enhanced petiole growth in both species. In R. palustris, accumulated ethylene stimulated elongation by inhibiting biosynthesis of ABA via a reduction of RpNCED expression and enhancing degradation of ABA to phaseic acid. Externally applied ABA inhibited petiole elongation and prevented the upregulation of gibberellin A(1) normally found in submerged R. palustris. In R. acetosa submergence did not stimulate petiole elongation nor did it depress levels of ABA. However, if ABA concentrations in R. acetosa were first artificially reduced, submergence (but not ethylene) was then able to enhance petiole elongation strongly. This result suggests that in Rumex a decrease in ABA is a prerequisite for ethylene and other stimuli to promote elongation.