Conformational Recognition of an Intrinsically Disordered Protein

There is a growing interest in understanding the properties of intrinsically disordered proteins (IDPs); however, the characterization of these states remains an open challenge. IDPs appear to have functional roles that diverge from those of folded proteins and revolve around their ability to act as hubs for protein-protein interactions. To gain a better understanding of the modes of binding of IDPs, we combined statistical mechanics, calorimetry, and NMR spectroscopy to investigate the recognition and binding of a fragment from the disordered protein Gab2 by the growth factor receptor-bound protein 2 (Grb2), a key interaction for normal cell signaling and cancer development. Structural ensemble refinement by NMR chemical shifts, thermodynamics measurements, and analysis of point mutations indicated that the population of preexisting bound conformations in the free-state ensemble of Gab2 is an essential determinant for recognition and binding by Grb2. A key role was found for transient polyproline II (PPII) structures and extended conformations. Our findings are likely to have very general implications for the biological behavior of IDPs in light of the evidence that a large fraction of these proteins possess a specific propensity to form PPII and to adopt conformations that are more extended than the typical random-coil states.     

Herbivore responses to nutrient enrichment and landscape heterogeneity in a mangrove ecosystem

Complex gradients in forest structure across the landscape of offshore mangrove islands in Belize are associated with nutrient deficiency and flooding. While nutrient availability can affect many ecological processes, here we investigate how N and P enrichment interact with forest structure in three distinct zones (fringe, transition, dwarf) to alter patterns of herbivory as a function of folivory, loss of yield, and tissue mining. The effects of nutrient addition and zone varied by functional feeding group or specific herbivore. Folivory ranged from 0 to 0.4% leaf area damaged per month, but rates did not vary by either nutrient enrichment or zone. Leaf lifetime damage ranged from 3 to 10% of the total leaf area and was caused primarily by the omnivorous tree crab Aratus pisonii. We detected two distinct spatial scales of response by A. pisonii that were unrelated to nutrient treatment, i.e., most feeding damage occurred in the fringe zone and crabs fed primarily on the oldest leaves in the canopy. Loss of yield caused by the bud moth Ecdytolopha sp. varied by zone but not by nutrient treatment. A periderm-mining Marmara sp. responded positively to nutrient enrichment and closely mirrored the growth response by Rhizophora mangle across the tree height gradient. In contrast, a leaf-mining Marmara sp. was controlled by parasitoids and predators that killed >89% of its larvae. Thus, nutrient availability altered patterns of herbivory of some but not all mangrove herbivores. These findings support the hypothesis that landscape heterogeneity of the biotic and abiotic environment has species-specific effects on community structure and trophic interactions. Predicting how herbivores respond to nutrient over-enrichment in mangrove ecosystems also requires an assessment of habitat heterogeneity coupled with feeding strategies and species-specific behavior measured on multiple scales of response.     

Convergence of molecular dynamics simulations of membrane proteins

The central question in evaluating almost any result from a molecular dynamics simulation is whether the calculation has converged. Unfortunately, assessing the ergodicity of a single trajectory is very difficult to do. In this work, we assess the sampling of molecular dynamics simulations of the membrane protein rhodopsin by comparing the results from 26 independent trajectories, each run for 100 ns. By examining principal components and cluster populations, we show that rhodopsin's fluctuations are not well described by 100 ns of dynamics, and that the sampling is not fully converged even for individual loops. The results serve as a reminder of the caution required when interpreting molecular dynamics simulations of macromolecules.     

Testing the growth rate vs. geochemical hypothesis for latitudinal variation in plant nutrients

Two hypotheses have been proposed to explain increases in plant nitrogen (N) and phosphorus (P) concentrations with latitude: (i) geochemical limitation to P availability in the tropics and (ii) temperature driven variation in growth rate, where greater growth rates (requiring greater nutrient levels) are needed to complete growth and reproduction within shorter growing seasons in temperate than tropical climates. These two hypotheses were assessed in one forest type, intertidal mangroves, using fertilized plots at sites between latitudes 36º S and 27º N. The N and P concentrations in mangrove leaf tissue increased with latitude, but there were no trends in N : P ratios. Growth rates of trees, adjusted for average minimum temperature showed a significant increase with latitude supporting the Growth Rate Hypothesis. However, support for the Geochemical Hypothesis was also strong; both photosynthetic P use efficiency and nutrient resorption efficiency decreased with increasing latitude, indicating that P was less limiting to metabolism at the higher latitudes. Our study supports the hypothesis that historically low P availability in the tropics has been an important selective pressure shaping the evolution of plant traits.     

Impact of Continuous Cropping of Lettuce on the Disease Dynamics of Bottom Rot and Genotypic Diversity of Rhizoctonia solani AG 1‐IB

The impact of continuous cropping of lettuce on the disease dynamics of bottom rot and genotypic diversity of the causal pathogen Rhizoctonia solani AG 1‐IB was studied over 3 years with two crops per year within a field naturally infested with R. solani the pathogen. This field had not had lettuce cultivated in it for 7 years. The disease incidence (DI) and disease severity (DS) were assessed at each harvest and mapped. Surprisingly, a high DI was already observed in the first crop of year one of this field study. In addition, the pathogen was also found to be evenly distributed. Severely infected plants occurred mainly in patches, and the position varied between crops. A significant increase in DS was already observed in the second year, and both temperature conditions and continuous cropping influenced the DS on average over time. Rhizoctonia isolates were randomly collected from the first crop in 1999 and the sixth crop in 2001. The genotypic diversity within the subgroup of R. solani AG 1‐IB was analysed by BOX‐PCR genomic fingerprinting and the aggressiveness of isolates by bioassay. The fingerprints revealed a high level of genotypic diversity within the AG 1‐IB field population. However, continuous cropping was found not to have an impact on genotypic diversity and aggressiveness.     

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E₂) are distinct from those in non-E₂-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E₂ proliferative H1793 and non-E₂-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E₂ or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.

     

Beyond DNA binding - a review of the potential mechanisms mediating quinacrine's therapeutic activities in parasitic infections,inflammation, and cancers

Background

Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin^-/-, integrin-beta1^-/-, focal adhesion kinase (FAK)^-/- and Src/Yes/Fyn^-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake.

Results

Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2^-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry.

Conclusion

Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.     

Self-organization and regulation of intrinsically disordered proteins with folded N-termini

How do mostly disordered proteins coordinate the specific assembly of very large signal transduction protein complexes? A newly emerging hypothesis may provide some clues towards a molecular mechanism.