Surface plasmon resonance analysis of the binding of high‐risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI‐1

The E6 oncoproteins from high‐risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain‐containing proteins. Human MAGI‐1 is a multi‐PDZ domain protein implicated into protein complex assembly at cell–cell contacts. High‐risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI‐1 via a C‐terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione‐S‐transferase (GST). This approach was applied to measure the binding of MAGI‐1 PDZ1 to the C‐termini of viral or cellular proteins. Both high‐risk mucosal HPV E6 C‐terminal peptides and cellular partners of MAGI‐1 PDZ1 bind to MAGI‐1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI‐1 PDZ1 shows a preference for C‐termini with a valine at position 0 and a negative charge at position ?3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site‐directed mutagenesis of the HPV16 C‐terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ‐binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K₄₉₉ residue of MAGI‐1 as a novel determinant of binding specificity. Finally, we showed that MAGI‐1 PDZ1 also binds to the C‐termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI‐1. Copyright © 2010 John Wiley & Sons, Ltd.     

A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency

A short G1 phase is a characteristic feature of mouse embryonic stem cells (ESCs). To determine if there is a causal relationship between G1 phase restriction and pluripotency, we made use of the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) reporter system to FACS-sort ESCs in the different cell cycle phases. Hence, the G1 phase cells appeared to be more susceptible to differentiation, particularly when ESCs self-renewed in the naïve state of pluripotency. Transitions from ground to naïve, then from naïve to primed states of pluripotency were associated with increased durations of the G1 phase, and cyclin E-mediated alteration of the G1/S transition altered the balance between self-renewal and differentiation. LIF withdrawal resulted in a lengthening of the G1 phase in naïve ESCs, which occurred prior to the appearance of early lineage-specific markers, and could be reversed upon LIF supplementation. We concluded that the short G1 phase observed in murine ESCs was a determinant of naïve pluripotency and was partially under the control of LIF signaling.     

Priming of calcium mobilization in human neutrophils by granulocyte-macrophage colony-stimulating factor: Evidence for an involvement of phospholipase D-derived phosphatidic acid

Human neutrophils pre-incubated with granulocyte-macrophage-colony-stimulating factor (GM-CSF) exhibit an enhanced mobilization of calcium in response to secondary stimuli such as chemotactic factors. The mechanisms underlying this priming effect of GM-CSF were examined. It was first demonstrated that the additional calcium mobilized by chemotactic factors in GM-CSF-treated cells was derived from intracellular stores and was associated neither with an increased permeability to calcium nor with production of inositol 1,4,5-trisphosphate. These results indicated that GM-CSF called upon a novel mechanism in order to enhance the mobilization of calcium in human neutrophils. The growth factor has recently been shown to prime phospholipase D leading to an enhanced activation by chemotactic factors and an augmented production of phosphatidic acid. Furthermore the ability of exogenous phosphatidic acid to mobilize calcium in cell types other than neutrophils has been previously demonstrated. Therefore, we examined the potential involvement of phospholipase D in the priming of the calcium response by GM-CSF in human neutrophils. Inhibition of the production of the fMet-Leu-Phe-stimulated production of phosphatidic acid by ethanol or wortmannin had only marginal effects on the concurrent mobilization of calcium. However, the priming of the mobilization of calcium by GM-CSF was greatly decreased in cells treated with either ethanol or wortmannin. These results provide strong support for the hypothesis that the production of phosphatidic acid, which is enhanced in GM-CSF-treated cells, is linked to an increased mobilization of intracellular calcium. These results may have relevance to the mechanism of action of GM-CSF in mature haematopoeitic cells as well to the mitogenic activity of other growth factors.     

Pseudo-nitzschia species population dynamics in the Quoddy Region, Bay of Fundy

The population dynamics of Pseudo-nitzschia species from the Quoddy Region of the Bay of Fundy was examined in a field-based study. Five stations were sampled over a period of 11 weeks during the course of one discrete bloom episode in 2003 with seven species of Pseudo-nitzschia found and enumerated: P. americana, P. delicatissima, P. pseudodelicatissima, P. fraudulenta, P. pungens, P. seriata and P. subpacifica. We related species abundance to physical and chemical properties of seawater (transparency, fluorescence, silicate, phosphate, nitrite + nitrate, ammonia, nitrite, oxygen, sigma-t, tidal level, tidal state and total depth of water column) using canonical correspondence analysis (CCA) to identify factors explaining the greatest amount of variance in their temporal and spatial distribution patterns. Our results indicate that abundance of species and groups of species correlated well with certain specific chemical and physical properties of seawater. P. pseudodelicatissima and P. delicatissima abundance was positively correlated with nitrates and P. americana with depth of the water column. P. pungens was more abundant in samples with higher concentrations of phosphates and lower concentrations of nitrates. P. seriata abundance was negatively associated with total fluorescence. P. subpacifica and P. fraudulenta abundances were not statistically related to any of the variables examined. Our data therefore provides direction for testable, hypothesis driven experiments that could provide predictive insights into the occurrence of certain harmful algal bloom (HAB) species should specific environmental variables be further affected along the gradients extracted in this study.     

Mhc polymorphisms fail to explain the heritability of phytohaemagglutinin-induced skin swelling in a wild passerine

Genetic estimates of the variability of immune responses are rarely examined in natural populations because of confounding environmental effects. As a result, and because of the difficulty of pinpointing the genetic determinants of immunity, no study has to our knowledge examined the contribution of specific genes to the heritability of an immune response in wild populations. We cross-fostered nestling house sparrows to disrupt the association between genetic and environmental effects and determine the heritability of the response to a classic immunological test, the phytohaemagglutinin (PHA)-induced skin swelling. We detected significant heritability estimates of the response to PHA, of body mass and tarsus length when nestlings were 5 and 10 days old. Variation at Mhc genes, however, did not explain a significant portion of the genetic variation of nestling swelling to PHA. Our results suggest that while PHA-induced swelling is influenced by the nest of origin, the importance of additive genetic variation relative to non-additive genetic variation and the genetic factors that influence the former in wild populations still need to be identified for this trait.     

E6 Proteins from Diverse Papillomaviruses Self-Associate Both InVitro and In Vivo

Papillomavirus E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here, we show that recombinant E6 proteins from eight human papillomavirus strains and one bovine papillomavirus strain exist as oligomeric and multimeric species. These species were characterized using a variety of biochemical and biophysical techniques, including analytical gel filtration, activity assays, surface plasmon resonance, electron microscopy and Fourier transform infrared spectroscopy. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein, which slows the formation of higher-order multimeric species. The proportion of each oligomeric form varies depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal human papillomavirus E6, retain binding to the ubiquitin ligase E6-associated protein and the capacity to degrade the proapoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process that is accompanied by a significant increase in the β-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggests that self-association is a general property of E6 proteins that occurs both in vitro and in vivo and might therefore be functionally relevant.     

Capturing protein-protein complexes at equilibrium: the holdup comparative chromatographic retention assay

The popular pulldown chromatographic assay detects complexes mediated by fusion proteins retained on affinity resin. The main limitation of this method is that it does not analyze complexes at equilibrium but after several washing steps. Consequently, fast-dissociating complexes may remain undetected. Here, we present the holdup assay, based on the principle of comparative chromatographic retention which eliminates the use of washing steps. The assay evaluates fractions of free and bound species at equilibrium. We used human papillomavirus oncoprotein E6, an E6-binding peptide and an E6-binding PDZ domain, to test several protocols utilizing pure proteins or expression extracts. The holdup assay is faster and more informative than the pulldown assay. It detects fast-dissociating complexes and it is also suited for evaluating equilibrium constants. It is potentially adaptable for automated determination of affinity constants and high-throughput analysis of interactions between proteins and other proteins, peptides, nucleic acids, or small regulatory molecules.