Use of amide exchange mass spectrometry to study conformational changes within the endopolygalacturonase II-homogalacturonan-polygalacturonase inhibiting protein system

Amide exchange mass spectrometry (MS) was used to study the enzyme endopolygalacturonase II (EPG-II) from Aspergillus niger as it binds to an oligosaccharide substrate. A localized decrease in the level of deuterium incorporation in EPG-II of the EPG-II-oligosaccharide complex relative to that of the free EPG-II identified the location of substrate contact, which is in agreement with published site specific mutation studies. In addition, when bound with substrate, regions of EPG-II remote from the substrate binding site became exposed to the solvent, as revealed by an increase in the amount of incorporated deuterium, indicating a conformational change in the enzyme. Fluorescence experiments were performed to provide additional evidence for an altered conformation of EPG-II as a result of substrate binding. This novel application of amide exchange-MS to the study of protein-carbohydrate binding has, for the first time, described in detail the conformational changes associated with EPG-II when it binds a substrate. Amide exchange-MS was also used to study the interactions of EPG-II and the polygalacturonase inhibitor protein (PGIP). Mass spectral data of the EPG-II-oligosaccharide complex in the presence of Phaseolus vulgaris PGIP indicate that the inhibitor contacts EPG-II at a site remote from the substrate binding cleft, and is restricting the conformational changes of EPG-II. Fluorescence experiments also revealed that upon binding of PGIP, the conformational changes mentioned above for the EPG-II-substrate complex are minimized. These results, together with previously reported data, point to a location on EPG-II for interaction with PGIP as well as a possible mechanism for noncompetitive inhibition of EPG-II.     

Proline-rich tyrosine kinase 2 (Pyk2) mediates vascular endothelial-cadherin-based cell-cell adhesion by regulating beta-catenin tyrosine phosphorylation

Vascular endothelial-cadherin (VE-cadherin) controls endothelial cell-cell adhesion and preserves endothelial integrity. In order to maintain endothelial barrier function, VE-cadherin function is tightly regulated through mechanisms that involve protein phosphorylation and cytoskeletal dynamics. Here, we show that loss of VE-cadherin function results in intercellular gap formation and a drop in electrical resistance of monolayers of primary human endothelial cells. Detailed analysis revealed that loss of endothelial cell-cell adhesion, induced by VE-cadherin-blocking antibodies, is preceded by and dependent on a rapid activation of Rac1 and increased production of reactive oxygen species. Moreover, VE-cadherin-associated beta-catenin is tyrosine-phosphorylated upon loss of cell-cell contact. Finally, the redox-sensitive proline-rich tyrosine kinase 2 (Pyk2) is activated and recruited to cell-cell junctions following the loss of VE-cadherin homotypic adhesion. Conversely, the inhibition of Pyk2 activity in endothelial cells by the expression of CRNK (CADTK/CAKbeta-related non-kinase), an N-terminal deletion mutant that acts in a dominant negative fashion, not only abolishes the increase in beta-catenin tyrosine phosphorylation but also prevents the loss of endothelial cell-cell contact. These results implicate Pyk2 in the reduced cell-cell adhesion induced by the Rac-mediated production of ROS through the tyrosine phosphorylation of beta-catenin. This signaling is initiated upon loss of VE-cadherin function and is important for our insight in the modulation of endothelial integrity.     

Altered distribution and co-localization of polycystin-2 with polycystin-1 in MDCK cells after wounding stress

Polycystin-1 and -2 are integral membrane glycoproteins defective in autosomal dominant polycystic kidney disease (ADPKD). Recent studies showed a coupled polycystin-1 and -2 action in cell signaling and channel activation suggesting an important biological role for the two proteins at the plasma membrane. To gain a better understanding about the (co)-distribution and dynamics of the polycystin-1 and -2 complex under stress conditions, we used a wound-healing model of Madine Darby canine kidney (MDCK) renal epithelial cells. In this model, cells near the wound edge undergo a process of reorganization to active migration, while cells further from the edge are unaffected and remain confluent. For the first time, endogenous polycystin-1 and -2 were found to partly co-localize in the plasma membrane of confluent monolayers, and both proteins co-localized in the primary cilium. Upon wound healing, the association of polycystin-2 to the membrane was greatly reduced at the wound edge and the submarginal cells. Polycystin-1 remained incorporated to the membrane at the edge of the cell sheet at all time points, although strongly reduced in lamellipodia-forming cells. Adherens junctions and desmosomes, and respective connected actin and keratin cytoskeleton were also disturbed in lamellipodia-forming cells. We propose that altered subcellular localization of polycystin-1 and -2 as a result of stress will affect signaling and other cellular processes mediated by these proteins.     

An oxo-ferryl tryptophan radical catalytic intermediate in cytochrome c and quinol oxidases trapped by microsecond freeze-hyperquenching (MHQ)

The pre-steady state reaction kinetics of the reduction of molecular oxygen catalyzed by fully reduced cytochrome oxidase from Escherichia coli and Paracoccus denitrificans were studied using the newly developed microsecond freeze-hyperquenching mixing-and-sampling technique. Reaction samples are prepared 60 and 200 micros after direct mixing of dioxygen with enzyme. Analysis of the reaction samples by low temperature UV-Vis spectroscopy indicates that both enzymes are trapped in the PM state. EPR spectroscopy revealed the formation of a mixture of two radicals in both enzymes. Based on its apparent g-value and lineshape, one of these radicals is assigned to a weakly magnetically coupled oxo-ferryl tryptophan cation radical. Implications for the catalytic mechanism of cytochrome oxidases are discussed.     

Is the trunk movement more perturbed after an asymmetric than after a symmetric perturbation during lifting?

Low back injury is associated with sudden movements and loading. Trunk motion after sudden loading depends on the stability of the spine prior to loading and on the trunk muscle activity in response to the loading. Both factors are not axis-symmetric. Therefore, it was hypothesized that the effects on trunk dynamics would be larger after an asymmetric than after a symmetric perturbation. Ten subjects lifted a crate in which, prior to lifting, a mass was displaced to the front or to the side without the subjects being aware of this. Crate and subject movements, crate reaction forces and muscle activity were recorded. From this, the stability prior to the perturbation was estimated, and the trunk angular kinematics and moments at the lumbo-sacral joint were calculated. Both perturbations only minimally affected the trunk kinematics, although the stability of the spine prior to the lifting movement was higher in the sagittal plane than in the frontal plane. In both conditions the stability appeared to be sufficient to absorb the applied perturbation.     

Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-Ad5 immunity

The high prevalence of pre-existing immunity to adenovirus serotype 5 (Ad5) in human populations may substantially limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for HIV-1 and other pathogens. A potential solution to this problem is to use vaccine vectors derived from adenovirus (Ad) serotypes that are rare in humans, such as Ad35. However, cross-reactive immune responses between heterologous Ad serotypes have been described and could prove a major limitation of this strategy. In particular, the extent of immunologic cross-reactivity between Ad5 and Ad35 has not previously been determined. In this study we investigate the impact of pre-existing anti-Ad5 immunity on the immunogenicity of candidate rAd5 and rAd35 vaccines expressing SIV Gag in mice. Anti-Ad5 immunity at levels typically found in humans dramatically blunted the immunogenicity of rAd5-Gag. In contrast, even high levels of anti-Ad5 immunity did not substantially suppress Gag-specific cellular immune responses elicited by rAd35-Gag. Low levels of cross-reactive Ad5/Ad35-specific CD4(+) T lymphocyte responses were observed, but were insufficient to suppress vaccine immunogenicity. These data demonstrate the potential utility of Ad35 as a candidate vaccine vector that is minimally suppressed by anti-Ad5 immunity. Moreover, these studies suggest that using Ad vectors derived from immunologically distinct serotypes may be an effective and general strategy to overcome the suppressive effects of pre-existing anti-Ad immunity.     

The amino terminus of Epstein-Barr Virus (EBV) nuclear antigen 1 contains AT hooks that facilitate the replication and partitioning of latent EBV genomes by tethering them to cellular chromosomes

During latency, Epstein-Barr virus (EBV) is stably maintained as a circular plasmid that is replicated once per cell cycle and partitioned at mitosis. Both these processes require a single viral protein, EBV nuclear antigen 1 (EBNA1), which binds two clusters of cognate binding sites within the latent viral origin, oriP. EBNA1 is known to associate with cellular metaphase chromosomes through chromosome-binding domains within its amino terminus, an association that we have determined to be required not only for the partitioning of oriP plasmids but also for their replication. One of the chromosome-binding domains of EBNA1 associates with a cellular nucleolar protein, EBP2, and it has been proposed that this interaction underlies that ability of EBNA1 to bind metaphase chromosomes. Here we demonstrate that EBNA1's chromosome-binding domains are AT hooks, a DNA-binding motif found in a family of proteins that bind the scaffold-associated regions on metaphase chromosomes. Further, we demonstrate that the ability of EBNA1 to stably replicate and partition oriP plasmids correlates with its AT hook activity and not its association with EBP2. Finally, we examine the contributions of EBP2 toward the ability of EBNA1 to associate with metaphase chromosomes in human cells, as well as support the replication and partitioning of oriP plasmids in human cells. Our results indicate that it is unlikely that EBP2 directly mediates these activities of EBNA1 in human cells.     

Common structure of rare replication-deficient E1-positive particles in adenoviral vector batches

The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 x 10(12) vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad.