Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation

Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide “stapling” to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR.     

3D collagen cultures under well‐defined dynamic strain: A novel strain device with a porous elastomeric support

The field of mechanobiology has grown tremendously in the past few decades, and it is now well accepted that dynamic stresses and strains can impact cell and tissue organization, cell–cell and cell–matrix communication, matrix remodeling, cell proliferation and apoptosis, cell migration, and many other cell behaviors in both physiological and pathophysiological situations. Natural reconstituted matrices like collagen and fibrin are often used for three‐dimensional (3D) mechanobiology studies because they naturally form fibrous architectures and are rich in cell adhesion sites; however, they are physically weak and typically contain >99% water, making it difficult to apply dynamic stresses to them in a truly 3D context. Here we present a composite matrix and strain device that can support natural matrices within a macroporous elastic structure of polyurethane. We characterize this system both in terms of its mechanical behavior and its ability to support the growth and in vivo‐like behaviors of primary human lung fibroblasts cultured in collagen. The porous polyurethane was created with highly interconnected pores in the hundreds of µm size scale, so that while it did not affect cell behavior in the collagen gel within the pores, it could control the overall elastic behavior of the entire tissue culture system. In this way, a well‐defined dynamic strain could be imposed on the 3D collagen and cells within the collagen for several days (with elastic recoil driven by the polyurethane) without the typical matrix contraction by fibroblasts when cultured in 3D collagen gels. We show lung fibroblast‐to‐myofibroblast differentiation under 30%, 0.1 Hz dynamic strain to validate the model and demonstrate its usefulness for a wide range of tissue engineering applications. Biotechnol. Bioeng. 2009;103: 217–225. © 2008 Wiley Periodicals, Inc.     

JNK signaling in insulin-producing cells is required for adaptive responses to stress in Drosophila

Adaptation to environmental challenges is critical for the survival of an organism. Repression of Insulin/IGF Signaling (IIS) by stress-responsive Jun-N-terminal Kinase (JNK) signaling is emerging as a conserved mechanism that allows reallocating resources from anabolic to repair processes under stress conditions. JNK activation in Insulin-producing cells (IPCs) is sufficient to repress Insulin and Insulin-like peptide (ILP) expression in rats and flies, but the significance of this interaction for adaptive responses to stress is unclear. In this study, it is shown that JNK activity in IPCs of flies is required for oxidative stress-induced repression of the Drosophila ILP2. It is found that this repression is required for growth adaptation to heat stress as well as adult oxidative stress tolerance, and that induction of stress response genes in the periphery is in part dependent on IPC-specific JNK activity. Endocrine control of IIS by JNK in IPCs is thus critical for systemic adaptation to stress.     

Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in disulfide bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti‐virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the disulfide bond forming enzymes, DsbB and VKOR, are required for Pseudomonas aeruginosa pathogenicity and Mycobacterium tuberculosis survival respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a disulfide‐bond sensitive β ‐galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development.     

Ancient papillomavirus-host co-speciation in Felidae

Background

Estimating evolutionary rates for slowly evolving viruses such as papillomaviruses (PVs) is not possible using fossil calibrations directly or sequences sampled over a time-scale of decades. An ability to correlate their divergence with a host species, however, can provide a means to estimate evolutionary rates for these viruses accurately. To determine whether such an approach is feasible, we sequenced complete feline PV genomes, previously available only for the domestic cat (Felis domesticus, FdPV1), from four additional, globally distributed feline species: Lynx rufus PV type 1, Puma concolor PV type 1, Panthera leo persica PV type 1, and Uncia uncia PV type 1.

Results

The feline PVs all belong to the Lambdapapillomavirus genus, and contain an unusual second noncoding region between the early and late protein region, which is only present in members of this genus. Our maximum likelihood and Bayesian phylogenetic analyses demonstrate that the evolutionary relationships between feline PVs perfectly mirror those of their feline hosts, despite a complex and dynamic phylogeographic history. By applying host species divergence times, we provide the first precise estimates for the rate of evolution for each PV gene, with an overall evolutionary rate of 1.95 × 10^-8 (95% confidence interval 1.32 × 10^-8 to 2.47 × 10^-8) nucleotide substitutions per site per year for the viral coding genome.

Conclusion

Our work provides evidence for long-term virus-host co-speciation of feline PVs, indicating that viral diversity in slowly evolving viruses can be used to investigate host species evolution. These findings, however, should not be extrapolated to other viral lineages without prior confirmation of virus-host co-divergence.     

Acidification effects on biofouling communities: winners and losers

How ocean acidification affects marine life is a major concern for science and society. However, its impacts on encrusting biofouling communities, that are both the initial colonizers of hard substrata and of great economic importance, are almost unknown. We showed that community composition changed significantly, from 92% spirorbids, 3% ascidians and 4% sponges initially to 47% spirorbids, 23% ascidians and 29% sponges after 100 days in acidified conditions (pH 7.7). In low pH, numbers of the spirorbid Neodexiospira pseudocorrugata were reduced ×5 compared to controls. The two ascidians present behaved differently with Aplidium sp. decreasing ×10 in pH 7.7, whereas Molgula sp. numbers were ×4 higher in low pH than controls. Calcareous sponge (Leucosolenia sp.) numbers increased ×2.5 in pH 7.7 over controls. The diatom and filamentous algal community was also more poorly developed in the low pH treatments compared to controls. Colonization of new surfaces likewise showed large decreases in spirorbid numbers, but numbers of sponges and Molgula sp. increased. Spirorbid losses appeared due to both recruitment failure and loss of existing tubes. Spirorbid tubes are comprised of a loose prismatic fabric of calcite crystals. Loss of tube materials appeared due to changes in the binding matrix and not crystal dissolution, as SEM analyses showed crystal surfaces were not pitted or dissolved in low pH conditions. Biofouling communities face dramatic future changes with reductions in groups with hard exposed exoskeletons and domination by soft‐bodied ascidians and sponges.     

Defining GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro models

GM-CSF and M-CSF (CSF-1) induce different phenotypic changes in macrophage lineage populations. The nature, extent, and generality of these differences were assessed by comparing the responses to these CSFs, either alone or in combination, in various human and murine macrophage lineage populations. The differences between the respective global gene expression profiles of macrophages, derived from human monocytes by GM-CSF or M-CSF, were compared with the differences between the respective profiles for macrophages, derived from murine bone marrow cells by each CSF. Only 17% of genes regulated differently by these CSFs were common across the species. Whether a particular change in relative gene expression is by direct action of a CSF can be confounded by endogenous mediators, such as type I IFN, IL-10, and activin A. Time-dependent differences in cytokine gene expression were noted in human monocytes treated with the CSFs; in this system, GM-CSF induced a more dramatic expression of IFN-regulated factor 4 (IRF4) than of IRF5, whereas M-CSF induced IRF5 but not IRF4. In the presence of both CSFs, some evidence of "competition" at the level of gene expression was observed. Care needs to be exercised when drawing definitive conclusions from a particular in vitro system about the roles of GM-CSF and M-CSF in macrophage lineage biology.     

Trinucleotide repeat containing 6a (Tnrc6a)-mediated microRNA function is required for development of yolk sac endoderm

Tnrc6 family members (Tnrc6a/b/c) are key components of the RNA-induced silencing complex in microRNA (miRNA)-mediated gene suppression. Here, we show that Tnrc6a, also known as GW182, is selectively expressed in the yolk sac endoderm and that gene trap disruption of GW182 leads to growth arrest and apoptosis. We found that targets of miRNAs highly expressed in the yolk sac are significantly derepressed in GW182(gt/gt) mutant mice, although levels of miRNAs are not altered. Specifically, growth arrest and apoptosis phenotype are associated with significant derepression of Cdkn1a (p21), Cdkn1c (P27), Lats1, Lats2, Rb1, Rbl, Bim, and Pten, known targets of miRNAs from miR-17/20/93/106 clusters highly expressed in yolk sac endoderm. Together, these data strongly suggest that GW182 is an essential functional component in the RNA-induced silencing complex for miRNA-mediated gene silencing in vivo, and selectively regulation of miRNA activity plays an important role in the proper development of yolk sac.