Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.     

Analysis of conditions affecting auto-phosphorylation of human kinases during expression in bacteria

Bacterial over-expression of kinases is often associated with high levels of auto-phosphorylation resulting in heterogeneous recombinant protein preparations or sometimes in insoluble protein. Here we present expression systems for nine kinases in Escherichia coli and, for the most heavily phosphorylated, the characterisation of factors affecting auto-phosphorylation. Experiments showed that the level of auto-phosphorylation was proportional to the rate of expression. Comparison of phosphorylation states following in vitro phosphorylation with phosphorylation states following expression in E. coli showed that the non-physiological 'hyper-phosphorylation' was occurring at sites that would require local unfolding to be accessible to a kinase active site. In contrast, auto-phosphorylation on unphosphorylated kinases that had been expressed in bacteria overexpressing λ-phosphatase was only observed on distinct exposed sites. Remarkably, the Ser/Thr kinase PLK4 auto-phosphorylated on a tyrosine residue (Tyr177) located in the activation segment. The results give support to a mechanism in which auto-phosphorylation occurs before or during protein folding. In addition, the expression systems and protocols presented will be a valuable resource to the research community.     

Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope

We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC′ loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC′ loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development.     

Bio-Logic Builder: A Non-Technical Tool for Building Dynamical,Qualitative Models

Computational modeling of biological processes is a promising tool in biomedical research. While a large part of its potential lies in the ability to integrate it with laboratory research, modeling currently generally requires a high degree of training in mathematics and/or computer science. To help address this issue, we have developed a web-based tool, Bio-Logic Builder, that enables laboratory scientists to define mathematical representations (based on a discrete formalism) of biological regulatory mechanisms in a modular and non-technical fashion. As part of the user interface, generalized “bio-logic” modules have been defined to provide users with the building blocks for many biological processes. To build/modify computational models, experimentalists provide purely qualitative information about a particular regulatory mechanisms as is generally found in the laboratory. The Bio-Logic Builder subsequently converts the provided information into a mathematical representation described with Boolean expressions/rules. We used this tool to build a number of dynamical models, including a 130-protein large-scale model of signal transduction with over 800 interactions, influenza A replication cycle with 127 species and 200+ interactions, and mammalian and budding yeast cell cycles. We also show that any and all qualitative regulatory mechanisms can be built using this tool.     

Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes

Proteomics is a valuable tool for establishing and comparing the protein content of defined tissues, cell types, or subcellular structures. Its use in non-model species is currently limited because the identification of peptides critically depends on sequence databases. In this study, we explored the potential of a preliminary cDNA database for the non-model species Pisum sativum created by a small number of massively parallel pyrosequencing (MPSS) runs for its use in proteomics and compared it to comprehensive cDNA databases from Medicago truncatula and Arabidopsis thaliana created by Sanger sequencing. Each database was used to identify proteins from a pea leaf chloroplast envelope preparation. It is shown that the pea database identified more proteins with higher accuracy, although the sequence quality was low and the sequence contigs were short compared to databases from model species. Although the number of identified proteins in non-species-specific databases could potentially be increased by lowering the threshold for successful protein identifications, this strategy markedly increases the number of wrongly identified proteins. The identification rate with non-species-specific databases correlated with spectral abundance but not with the predicted membrane helix content, and strong conservation is necessary but not sufficient for protein identification with a non-species-specific database. It is concluded that massively parallel sequencing of cDNAs substantially increases the power of proteomics in non-model species.     

Effects of environmental humidity on the survival and development of pine caterpillars, Dendrolimus tabulaeformis (Lepidoptera: Lasiocampidae)

The pine caterpillar, Dendrolimus tabulaeformis is one of the most important pests on Pinus tabulaeformis and other pine species in North China. In the present study, effects of relative humidity (RH) on the development and survival of pine caterpillars and soil moisture (SM) on their diapausing larvae were investigated. Low RH (20%) deferred the development of eggs and larvae, reduced egg hatching and larval surviving compared to 40%, 60% and 80% RH. Both low (20%) and high (100%) RH reduced egg hatching, but only 20% RH deferred the development of larvae, prolonged developmental duration and reduced the body mass and body length of larvae. The SM influenced the survival of diapausing larvae significantly. The dry treatment significantly reduced the supercooling points (SCPs), whereas increased the mortality and reduced body mass from 56.9 to 36.5 mg and body water content from 78% to 63% after 2 weeks' exposure. Therefore, higher RH is more favorable for the development of early instars and survival of diapausing larvae of the pine caterpillars.