Aggregation-Prone Motifs in Human Immunoglobulin G

Therapeutic antibodies of many different IgG subclasses (IgG1, IgG2 and IgG4) are used in the treatment of various cancers, rheumatoid arthritis and other inflammatory and infectious diseases. These antibodies are stored for long durations under high concentrations as required in the disease treatment. Unfortunately, these antibodies aggregate under these storage conditions, leading to a decrease in antibody activity and raising concerns about causing an immunological response. Thus, there is a tremendous need to identify the aggregation-prone regions in different classes of antibodies. We use the SAP (spatial-aggregation-propensity) technology based on molecular simulations to determine the aggregation-prone motifs in the constant regions of IgG1 classes of antibodies. Mutations engineered on these aggregation-prone motif regions led to antibodies of enhanced stability. Fourteen aggregation-prone motifs are identified, with each motif containing one to seven residues. While some of these motifs contain residues that are neighbors in primary sequence, others contain residues that are far apart in primary sequence but are close together in the tertiary structure. Comparison of the IgG1 sequence with those of other subclasses (IgG2, IgG3 and IgG4) showed that these aggregation-prone motifs are largely preserved among all IgG subclasses. Other broader classes of antibodies (IgA1, IgD, IgE and IgM), however, differed in these motif regions. The aggregation-prone motifs identified were therefore common to all IgG subclasses, but differ from those of non-IgG classes. Moreover, since the motifs identified are in the constant regions, they are applicable for all antibodies within the IgG class irrespective of the variable region. Thus, the motif regions identified could be modified on all IgGs to yield antibodies of enhanced stability.     

Thermodynamic constraints on the utility of ecological stoichiometry for explaining global biogeochemical patterns

Carbon and nitrogen cycles are coupled through both stoichiometric requirements for microbial biomass and dissimilatory metabolic processes in which microbes catalyse reduction‐oxidation reactions. Here, we integrate stoichiometric theory and thermodynamic principles to explain the commonly observed trade‐off between high nitrate and high organic carbon concentrations, and the even stronger trade‐off between high nitrate and high ammonium concentrations, across a wide range of aquatic ecosystems. Our results suggest these relationships are the emergent properties of both microbial biomass stoichiometry and the availability of terminal electron acceptors. Because elements with multiple oxidation states (i.e. nitrogen, manganese, iron and sulphur) serve as both nutrients and sources of chemical energy in reduced environments, both assimilative demand and dissimilatory uses determine their concentrations across broad spatial gradients. Conceptual and quantitative models that integrate rather than independently examine thermodynamic, stoichiometric and evolutionary controls on biogeochemical cycling are essential for understanding local to global biogeochemical patterns.     

The NT-26 cytochrome c552 and its role in arsenite oxidation

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c₅₅₂, is similar to a number of c-type cytochromes from the α-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c₅₅₂ revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.     

Nitrogen Dynamics in Ice Storm-Damaged Forest Ecosystems: Implications for Nitrogen Limitation Theory

Despite the widely recognized importance of disturbance in accelerating the loss of elements from land, there have been few empirical studies of the effects of natural disturbances on nitrogen (N) dynamics in forest ecosystems. We were provided the unusual opportunity for such study, partly because the intensively monitored watersheds at the Hubbard Brook Experimental Forest (HBEF), New Hampshire, experienced severe canopy damage following an ice storm. Here we report the effects of this disturbance on internal N cycling and loss for watershed 1 (W1) and watershed 6 (W6) at the HBEF and patterns of N loss from nine other severely damaged watersheds across the southern White Mountains. This approach allowed us to test one component of N limitation theory, which suggests that N losses accompanying natural disturbances can lead to the maintenance of N limitation in temperate zone forest ecosystems. Prior to the ice storm, fluxes of nitrate (NO₃ ^–) at the base of W1 and W6 were similar and were much lower than N inputs in atmospheric deposition. Following the ice storm, drainage water NO₃ ^– concentrations increased to levels that were seven to ten times greater than predisturbance values. We observed no significant differences in N mineralization, nitrification, or denitrification between damaged and undamaged areas in the HBEF watersheds, however. This result suggests that elevated NO₃ ^- concentrations were not necessarily due to accelerated rates of N cycling by soil microbes but likely resulted from decreased plant uptake of NO₃ ^-. At the regional scale, we observed high variability in the magnitude of NO₃ ^- losses: while six of the surveyed watersheds showed accelerated rates of NO₃ ^– loss, three did not. Moreover, in contrast to the strong linear relationship between NO₃ ^– loss and crown damage within HBEF watersheds [r ²: (W1 = 0.91, W6 = 0.85)], stream water NO₃ ^– concentrations were weakly related to crown damage (r ² = 0.17) across our regional sites. The efflux of NO₃ ^– associated with the ice storm was slightly higher than values reported for soil freezing and insect defoliation episodes, but was approximately two to ten times lower than NO₃ ^– fluxes associated with forest harvesting. Because over one half of the entire years worth of N deposition was lost following the ice storm, we conclude that catastrophic disturbances contribute synergistically to the maintenance of N limitation and widely observed delays of N saturation in northern, temperate zone forest ecosystems. Present address: Department of Ecology and Evolutionary Biology, Princeton University, Guyot Hall, Princeton, New Jersey 08544, USA.     

Poliovirus and its cellular receptor: a molecular genetic dissection of a virus/receptor affinity interaction

The ability of a virus to attach to a susceptible host cell is of utmost importance for the initiation of viral life cycle. Cell surface proteins called viral receptors mediate the initial steps of virus attachment and uptake. Poliovirus (PV) is one of the most studied animal viruses and its interaction with its cellular receptor, the human poliovirus receptor (hPVR) has been well characterized. This review will present our current understanding of the PV/hPVR interaction at the genetic and biochemical level. In addition, we will also discuss the implications of the PV/hPVR interaction on PV tissue tropism and the evolution of the three PV serotypes.     

Functional reconstitution of human neuropeptide Y (NPY) Y(2) and Y(4) receptors in Sf9 insect cells

The four functionally expressed human neuropeptide Y receptor subtypes (hY(1)R, hY(2)R, hY(4)R, hY(5)R) belong to class A of the G-protein-coupled receptors (GPCRs) and interact with pertussis toxin-sensitive G(i/o)-proteins. The number of small molecules described as ligands for hY(1)R and hY(5)R exceeds by far those for hY(2)R. Potent non-peptidergic ligands for the hY(4)R are not available so far. Here, we report on the functional reconstitution of the hY(2)R and the hY(4)R in Sf9 insect cells using the baculovirus system. Sf9 cells were genetically engineered by infection with up to four different baculoviruses, combining the receptors with G-proteins of the G(i/o) family and regulators of G-protein signaling (RGS) proteins to improve signal-to-noise ratio. In steady-state GTPase assays, using pNPY (Y(2)) and hPP (Y(4)), the GPCRs coupled to various G(i)/G(o)-proteins and both, RGS4 and GAIP, enhanced the signals. Co-expression systems hY(2)R + G?(i2) and hY(4)R + G?(i2)/G?(o) + RGS4, combined with G?(1)?(2), yielded best signal-to-noise ratios. hY(2)R function was validated using both agonistic peptides (NPY, PYY, NPY(13?36)) and selective non-peptidergic antagonists (BIIE0246 and derivatives), whereas the hY(4)R model was characterized with peptidergic agonists (PP, NPY, GW1229, and BW1911U90). Tunicamycin inhibited receptor N-glycosylation diminished NPY signals at hY(2)R and abolished hY(4)R function. Investigations with monovalent salts showed sensitivity of hY(4)R toward Na(+), revealing moderate constitutive activity. After validation, an acylguanidine (UR-PI284) was identified as a weak non-peptide Y(4)R antagonist. In summary, the established steady-state GTPase assays provide sensitive test systems for the characterization of Y(2) and Y(4) receptor ligands.     

The state of the art in the analysis of two-dimensional gel electrophoresis images

Software-based image analysis is a crucial step in the biological interpretation of two-dimensional gel electrophoresis experiments. Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments. We cover the process starting with the imaging of 2-D gels, quantitation of spots, creation of expression profiles to statistical expression analysis followed by the presentation of results. Challenges for analysis software as well as good practices are highlighted. We emphasize image warping and related methods that are able to overcome the difficulties that are due to varying migration positions of spots between gels. Spot detection, quantitation, normalization, and the creation of expression profiles are described in detail. The recent development of consensus spot patterns and complete expression profiles enables one to take full advantage of statistical methods for expression analysis that are well established for the analysis of DNA microarray experiments. We close with an overview of visualization and presentation methods (proteome maps) and current challenges in the field. An erratum to this article can be found at     

Mapping of interactions between human macrophages and Staphylococcus aureus reveals an involvement of MAP kinase signaling in the host defense

Staphylococcus aureus is a dangerous opportunistic human pathogen that causes serious invasive diseases when it reaches the bloodstream. Recent studies have shown that S. aureus is highly resistant to killing by professional phagocytes and that such cells even provide a favorable environment for intracellular survival of S. aureus. Importantly, the reciprocal interactions between phagocytes and S. aureus have remained largely elusive. Here we have employed kinase profiling to define the nature and time resolution of the human THP-1 macrophage response toward S. aureus and proteomics to identify the response of S. aureus toward macrophages. The results of these studies reveal major macrophage signaling pathways triggered by S. aureus and proteomic signatures of the responses of S. aureus to macrophages. We also identify human proteins bound to S. aureus that have potential roles in bacterial killing and internalization. Most noticeably, our observations challenge the classical concept that macrophage responses are mainly mediated through Toll-like receptor 2 and NF-κB signaling and highlight the important role of the stress-activated MAP kinase signaling in orchestrating the host defense.     

The human mineralocorticoid receptor only partially differentiates between different ligands after expression in fission yeast

Cardiac failure is a major health problem with increasing incidence due to aging of the population. Studies in both experimental animals and humans have suggested that aldosterone excess may have deleterious effects on cardiac function. In order to generate a novel screening system for the identification of aldosterone antagonists, we expressed the human mineralocorticoid receptor (MR) and the human glucocorticoid receptor (GR), respectively, in the fission yeast Schizosaccharomyces pombe. Reporter plasmids containing two hormone-responsive elements upstream of a fission yeast minimal promotor and either a lacZ gene (for quantification) or a neomycin gene (for survival screening) were constructed and cotransformed into fission yeast strains with expression plasmids for MR or GR. The functionality of the reporter systems was then tested using physiological ligands of both receptors as well as known inhibitors. Transactivating activity of MR could be stimulated by aldosterone, 11-deoxycorticosterone, 11-deoxycortisol, cortisol, cortisone, and spironolactone, but not by progesterone, while GR activity was stimulated by cortisol and cortisone, but also not by progesterone. Taken together, we have succeeded in establishing fission yeast-based screening systems that allow the identification of MR- or GR-interacting compounds. Moreover, our data show that after expression in fission yeast both receptors did not differentiate between steroids with different configurations at positions 11beta, 17 and 18. This finding suggests that only recognition of C-21 substituents may be accomplished by the receptor proteins alone, while the physiologically important selectivity towards other positions of the steroid ligand depends on other factors which are not conserved from fission yeast to man.