An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins

Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, coimmunoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system.     

Multiple Wnts and frizzled receptors regulate anteriorly directed cell and growth cone migrations in Caenorhabditis elegans

A set of conserved molecules guides axons along the metazoan dorsal-ventral axis. Recently, Wnt glycoproteins have been shown to guide axons along the anterior-posterior (A/P) axis of the mammalian spinal cord. Here, we show that, in the nematode Caenorhabditis elegans, multiple Wnts and Frizzled receptors regulate the anterior migrations of neurons and growth cones. Three Wnts are expressed in the tail, and at least one of these, EGL-20, functions as a repellent. We show that the MIG-1 Frizzled receptor acts in the neurons and growth cones to promote their migrations and provide genetic evidence that the Frizzleds MIG-1 and MOM-5 mediate the repulsive effects of EGL-20. While these receptors mediate the effects of EGL-20, we find that the Frizzled receptor LIN-17 can antagonize MIG-1 signaling. Our results indicate that Wnts play a key role in A/P guidance in C. elegans and employ distinct mechanisms to regulate different migrations.     

Two domains of the erythropoietin receptor are sufficient for Jak2 binding/activation and function

Biochemical and genetic studies have shown that Jak2 is an essential component of EpoR signal transduction which is required for normal erythropoiesis. However, whether Jak2 is the sole direct mediator of EpoR signal transduction remains controversial. To address this issue, we have used an extensive and systematic mutational analysis across the EpoR cytoplasmic tail and transmembrane domain with the goal of determining whether mutants that negatively affected EpoR biological activity but retained Jak2 activation could be identified. Analysis of over 40 mutant receptors established that two large domains in the membrane-proximal region, which include the previously defined Box1 and Box2 domains as well as a highly conserved glycine among cytokine receptors, are required for Jak2 binding and activation and to sustain biological activity of the receptor. Importantly, none of the mutants that lost the ability to activate Jak2 retained the ability to bind Jak2, thus questioning the validity of models of receptor reorientation for Jak2 activation. Also, no correlation was made between cell surface expression of the receptor and its ability to bind Jak2, thus questioning the role of Jak2 in trafficking the receptor to the plasma membrane. Collectively, the results suggest that Jak2 is the sole direct signaling molecule downstream of EpoR required for biological activity.     

A methodology for evaluating biocide release rate, surface roughness and leach layer formation in a TBT-free, self-polishing antifouling coating

Due to the forthcoming IMO ban on the use of tributyltin (TBT) antifouling paints, a new generation of TBT-free coatings has been developed that typically contain cuprous oxide and an organic co-biocide. Accurate and reproducible test methods are needed to evaluate the performance and environmental impact of these new coatings. This study investigated a methodology for evaluating TBT-free, AF coatings containing cuprous oxide. A commercially available AF coating underwent rotary immersion testing at 0, 0.51 and 2.05 m s-1. Scanning electron microscopy (SEM) and energy dispersive x-ray (EDX) analysis were used to assess leach layer formation, percentage cuprous oxide by weight and particle size distribution (PSD). Biocide release rates and surface roughness were also measured. An increase in rotary speed caused a spike in Cu2+ release rate after which the release rate stabilised to previous levels. An increase in leach layer thickness was also observed after the rotary speed increase. A model is suggested to account for the observations.     

Can markers injected into a single-loop anterior cruciate ligament graft define the axes of the tibial and femoral tunnels? A cadaveric study using roentgen stereophotogrammetric analysis

Lengthening of a soft-tissue anterior cruciate ligament (ACL) graft construct over time, which leads to an increase in anterior laxity following ACL reconstruction, can result from relative motions between the graft and fixation devices and between the fixation devices and bone. To determine these relative motions using Roentgen stereophotogrammetry (RSA), it is first necessary to identify the axes of the tibial and femoral tunnels. The purpose of this in vitro study was to determine the error in using markers injected into the portions of a soft-tissue tendon graft enclosed within the tibial and femoral tunnels to define the axes of these tunnels. Markers were injected into the tibia, femur, and graft in six cadaveric legs the knees of which were reconstructed with single-loop tibialis grafts. The axes of the tunnels were defined by marker pairs that were injected into the bones on lines parallel to the walls of the tibial and femoral tunnels (i.e., standard). By using marker pairs injected into the portions of the graft enclosed within the tibial and femoral tunnels and the marker pairs aligned with the tunnel axes, the directions of vectors were determined by using RSA, while a 150 N anterior force was transmitted at the knee. The average and standard deviations of the angle between the two vectors were 5.5+/-3.3 deg. This angle translates into an average error and standard deviation of the error in lengthening quantities (i.e., relative motions along the tunnel axes) at the sites of fixation of (0.6+/-0.8)%. Identifying the axes of the tunnels by using marker pairs in the graft rather than marker pairs in the walls of the tunnels will shorten the surgical procedure by eliminating the specialized tools and time required to insert marker pairs in the tunnel walls and will simplify the data analysis in in vivo studies.     

Substitution of a residue contacting the triphosphate moiety of the incoming nucleotide increases the fidelity of yeast DNA polymerase zeta

DNA polymerase zeta (pol zeta), which is required for DNA damage-induced mutagenesis, functions in the error-prone replication of a wide range of DNA lesions. During this process, pol zeta extends from nucleotides incorporated opposite template lesions by other polymerases. Unlike classical polymerases, pol zeta efficiently extends from primer-terminal base pairs containing mismatches or lesions, and it synthesizes DNA with moderate fidelity. Here we describe genetic and biochemical studies of three yeast pol zeta mutant proteins containing substitutions of highly conserved amino acid residues that contact the triphosphate moiety of the incoming nucleotide. The R1057A and K1086A proteins do not complement the rev3Delta mutation, and these proteins have significantly reduced polymerase activity relative to the wild-type protein. In contrast, the K1061A protein partially complements the rev3Delta mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased fidelity relative to wild-type pol zeta and is somewhat less efficient at extending from mismatched primer-terminal base pairs. These findings have important implications both for the evolutionary divergence of pol zeta from classical polymerases and for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions.     

Two highly related regulatory subunits of PP2A exert opposite effects on TGF-beta/Activin/Nodal signalling

We identify Balpha (PPP2R2A) and Bdelta (PPP2R2D), two highly related members of the B family of regulatory subunits of the protein phosphatase PP2A, as important modulators of TGF-beta/Activin/Nodal signalling that affect the pathway in opposite ways. Knockdown of Balpha in Xenopus embryos or mammalian tissue culture cells suppresses TGF-beta/Activin/Nodal-dependent responses, whereas knockdown of Bdelta enhances these responses. Moreover, in Drosophila, overexpression of Smad2 rescues a severe wing phenotype caused by overexpression of the single Drosophila PP2A B subunit Twins. We show that, in vertebrates, Balpha enhances TGF-beta/Activin/Nodal signalling by stabilising the basal levels of type I receptor, whereas Bdelta negatively modulates these pathways by restricting receptor activity. Thus, these highly related members of the same subfamily of PP2A regulatory subunits differentially regulate TGF-beta/Activin/Nodal signalling to elicit opposing biological outcomes.     

Lung-selective gene responses to alveolar hypoxia: potential role for the bone morphogenetic antagonist gremlin in pulmonary hypertension

Pulmonary hypoxia is a common complication of chronic lung diseases leading to the development of pulmonary hypertension. The underlying sustained increase in vascular resistance in hypoxia is a response unique to the lung. Thus we hypothesized that there are genes for which expression is altered selectively in the lung in response to alveolar hypoxia. Using a novel subtractive array strategy, we compared gene responses to hypoxia in primary human pulmonary microvascular endothelial cells (HMVEC-L) with those in cardiac microvascular endothelium and identified 90 genes (forming 9 clusters) differentially regulated in the lung endothelium. From one cluster, we confirmed that the bone morphogenetic protein (BMP) antagonist, gremlin 1, was upregulated in the hypoxic murine lung in vivo but was unchanged in five systemic organs. We also demonstrated that gremlin protein was significantly increased by hypoxia in vivo and inhibited HMVEC-L responses to BMP stimulation in vitro. Furthermore, significant upregulation of gremlin was measured in lungs of patients with pulmonary hypertensive disease. From a second cluster, we showed that CXC receptor 7, a receptor for the proangiogenic chemokine CXCL12, was selectively upregulated in the hypoxic lung in vivo, confirming that our subtractive strategy had successfully identified a second lung-selective hypoxia-responsive gene. We conclude that hypoxia, typical of that encountered in pulmonary disease, causes lung-specific alterations in gene expression. This gives new insights into the mechanisms of pulmonary hypertension and vascular loss in chronic lung disease and identifies gremlin 1 as a potentially important mediator of vascular changes in hypoxic pulmonary hypertension.     

Differential accumulation of retroelements and diversification of NB-LRR disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean

The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.