On interpretation of protein X‐ray structures: Planarity of the peptide unit

Pauling's mastery of peptide stereochemistry—based on small molecule crystal structures and the theory of chemical bonding—led to his realization that the peptide unit is planar and then to the Pauling–Corey–Branson model of the α‐helix. Similarly, contemporary protein structure refinement is based on experimentally determined diffraction data together with stereochemical restraints. However, even an X‐ray structure at ultra‐high resolution is still an under‐determined model in which the linkage among refinement parameters is complex. Consequently, restrictions imposed on any given parameter can affect the entire structure. Here, we examine recent studies of high resolution protein X‐ray structures, where substantial distortions of the peptide plane are found to be commonplace. Planarity is assessed by the ω‐angle, a dihedral angle determined by the peptide bond (C? N) and its flanking covalent neighbors; for an ideally planar trans peptide, ω = 180°. By using a freely available refinement package, Phenix [Afonine et al. (2012) Acta Cryst. D, 68:352–367], we demonstrate that tightening default restrictions on the ω‐angle can significantly reduce apparent deviations from peptide unit planarity without consequent reduction in reported evaluation metrics (e.g., R‐factors). To be clear, our result does not show that substantial non‐planarity is absent, only that an equivalent alternative model is possible. Resolving this disparity will ultimately require improved understanding of the deformation energy. Meanwhile, we urge inclusion of ω‐angle statistics in new structure reports in order to focus critical attention on the usual practice of assigning default values to ω‐angle constraints during structure refinement. Proteins 2015; 83:1687–1692. © 2015 Wiley Periodicals, Inc.     

Palaeontology: the 165-million-year itch

New flea-like fossils from China provide a rare, tantalizing glimpse of bizarre insects in the Cretaceous and Jurassic. Possibly the oldest flea-like animals known, they provide a challenge to the functional morphologist to infer which animals they may have targeted.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

Structural role of RKS motifs in chromatin interactions: a molecular dynamics study of HP1 bound to a variably modified histone tail

The current understanding of epigenetic signaling assigns a central role to post-translational modifications that occur in the histone tails. In this context, it has been proposed that methylation of K9 and phosphorylation of S10 in the tail of histone H3 represent a binary switch that controls its reversible association to heterochromatin protein 1 (HP1). To test this hypothesis, we performed a comprehensive molecular dynamics study in which we analyzed a crystallographically defined complex that involves the HP1 chromodomain and an H3 tail peptide. Microsecond-long simulations show that the binding of the trimethylated K9 H3 peptide in the aromatic cage of HP1 is only slightly affected by S10 phosphorylation, because the modified K9 and S10 do not interact directly with one another. Instead, the phosphate group of S10 seems to form a persistent intramolecular salt bridge with R8, an interaction that can provoke a major structural change and alter the hydrogen-bonding regime in the H3-HP1 complex. These observations suggest that interactions between adjacent methyl-lysine and phosphoserine side chains do not by themselves provide a binary switch in the H3-HP1 system, but arginine-phosphoserine interactions, which occur in both histones and nonhistone proteins in the context of a conserved RKS motif, are likely to serve a key regulatory function.     

Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca2+ signaling in dystrophic mdx mouse muscle

The majority of the skeletal muscle plasma membrane is internalized as part of the tubular (t-) system, forming a standing junction with the sarcoplasmic reticulum (SR) membrane throughout the muscle fiber. This arrangement facilitates not only a rapid and large release of Ca(2+) from the SR for contraction upon excitation of the fiber, but has also direct implications for other interdependent cellular regulators of Ca(2+). The t-system plasma membrane Ca-ATPase (PMCA) and store-operated Ca(2+) entry (SOCE) can also be activated upon release of SR Ca(2+). In muscle, the SR Ca(2+) sensor responsible for rapidly activated SOCE appears to be the stromal interacting molecule 1L (STIM1L) isoform of STIM1 protein, which directly interacts with the Orai1 Ca(2+) channel in the t-system. The common isoform of STIM1 is STIM1S, and it has been shown that STIM1 together with Orai1 in a complex with the partner protein of STIM (POST) reduces the activity of the PMCA. We have previously shown that Orai1 and STIM1 are upregulated in dystrophic mdx mouse muscle, and here we show that STIM1L and PMCA are also upregulated in mdx muscle. Moreover, we show that the ratios of STIM1L to STIM1S in wild-type (WT) and mdx muscle are not different. We also show a greater store-dependent Ca(2+) influx in mdx compared with WT muscle for similar levels of SR Ca(2+) release while normal activation and deactivation properties were maintained. Interestingly, the fiber-averaged ability of WT and mdx muscle to extrude Ca(2+) via PMCA was found to be the same despite differences in PMCA densities. This suggests that there is a close relationship among PMCA, STIM1L, STIM1S, Orai1, and also POST expression in mdx muscle to maintain the same Ca(2+) extrusion properties as in the WT muscle.     

Meta-analysis of genome-wide scans for total body BMD in children and adults reveals allelic heterogeneity and age-specific effects at the WNT16 locus

To identify genetic loci influencing bone accrual, we performed a genome-wide association scan for total-body bone mineral density (TB-BMD) variation in 2,660 children of different ethnicities. We discovered variants in 7q31.31 associated with BMD measurements, with the lowest P = 4.1 × 10(-11) observed for rs917727 with minor allele frequency of 0.37. We sought replication for all SNPs located ± 500 kb from rs917727 in 11,052 additional individuals from five independent studies including children and adults, together with de novo genotyping of rs3801387 (in perfect linkage disequilibrium (LD) with rs917727) in 1,014 mothers of children from the discovery cohort. The top signal mapping in the surroundings of WNT16 was replicated across studies with a meta-analysis P = 2.6 × 10(-31) and an effect size explaining between 0.6%-1.8% of TB-BMD variance. Conditional analyses on this signal revealed a secondary signal for total body BMD (P = 1.42 × 10(-10)) for rs4609139 and mapping to C7orf58. We also examined the genomic region for association with skull BMD to test if the associations were independent of skeletal loading. We identified two signals influencing skull BMD variation, including rs917727 (P = 1.9 × 10(-16)) and rs7801723 (P = 8.9 × 10(-28)), also mapping to C7orf58 (r(2) = 0.50 with rs4609139). Wnt16 knockout (KO) mice with reduced total body BMD and gene expression profiles in human bone biopsies support a role of C7orf58 and WNT16 on the BMD phenotypes observed at the human population level. In summary, we detected two independent signals influencing total body and skull BMD variation in children and adults, thus demonstrating the presence of allelic heterogeneity at the WNT16 locus. One of the skull BMD signals mapping to C7orf58 is mostly driven by children, suggesting temporal determination on peak bone mass acquisition. Our life-course approach postulates that these genetic effects influencing peak bone mass accrual may impact the risk of osteoporosis later in life.     

Model-guided fieldwork: practical guidelines for multidisciplinary research on wildlife ecological and epidemiological dynamics

Infectious disease ecology has recently raised its public profile beyond the scientific community due to the major threats that wildlife infections pose to biological conservation, animal welfare, human health and food security. As we start unravelling the full extent of emerging infectious diseases, there is an urgent need to facilitate multidisciplinary research in this area. Even though research in ecology has always had a strong theoretical component, cultural and technical hurdles often hamper direct collaboration between theoreticians and empiricists. Building upon our collective experience of multidisciplinary research and teaching in this area, we propose practical guidelines to help with effective integration among mathematical modelling, fieldwork and laboratory work. Modelling tools can be used at all steps of a field-based research programme, from the formulation of working hypotheses to field study design and data analysis. We illustrate our model-guided fieldwork framework with two case studies we have been conducting on wildlife infectious diseases: plague transmission in prairie dogs and lyssavirus dynamics in American and African bats. These demonstrate that mechanistic models, if properly integrated in research programmes, can provide a framework for holistic approaches to complex biological systems.     

Interplay between oncogenic K-Ras and wild-type H-Ras in Caco2 cell transformation

Mutation of RAS genes is one of the most common oncogenic alterations in cancer and acquisition of activating RAS mutations has been demonstrated to cause progression of colorectal adenoma to cancer. The aim of this study was to identify changes in the proteome of the intermediate-stage colorectal cancer cell line Caco2, induced by ectopic expression of two distinct RAS proteins, KRAS(V12) and HRAS(V12), in their mutated, constitutively active form. Using 2D-gel electrophoresis, followed by LC-MS/MS we identified almost 200 differentially expressed proteins in pair-wise comparisons of Caco2 vs Caco2-KRAS(V12) and Caco2 vs Caco2-HRAS(V12). Although many of the affected proteins were unique for each pair, there were also substantial similarities. Interestingly, transformation by the mutant KRAS(V12) gene resulted in elevated expression levels and activity of endogenous H-ras protein. Silencing the latter with a specific RNAi reversed several proteomic changes observed in KRAS(V12)-transformed cells, suggesting that oncogenic K-ras partly exerts its effects through endogenous H-ras activation. Alterations in the expression of cytoskeletal and cell adhesion proteins, caused by HRAS siRNA treatment, correlated with a reduction in the invasive properties of Caco2-KRAS(V12) cells. Our data suggest a novel interplay between K-ras and H-ras, with possible implications for colorectal carcinogenesis.     

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.