Peptide Finder: mapping measured molecular masses to peptides and proteins

The identification of unknown amino acid sequences of peptides as well as protein identification is of great significance in proteomics. Here, we present a publicly available web application that facilitates a high resolution mapping of measured molecular masses to peptides and proteins, irrespectively of the enzyme/digestion method used. Furthermore, multi-filtering may be applied in terms of measured mass tolerance, molecular mass and isoelectric point range as well as pattern matching to refine the results. This approach serves complementary to the existing solutions for protein identification and gives insights in novel peptides discovery and protein identification at the cases where the identification scores from the other approaches may be below significance threshold. Peptide Finder has been proven useful in proteomics procedures with experimental data from MALDI-TOF. AVAILABILITY: Peptide Finder web-application is available at http://bioserver-1.bioacademy.gr/Bioserver/PeptideFinder/.     

Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca²⁺ and Mn²⁺. Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.Anthrax is an infectious disease caused by the encapsulated, spore-forming bacterium Bacillus anthracis. Systemic forms of the disease, such as inhalational anthrax, are characterized by nonspecific early symptoms, rapid progression, and lethality approaching 100% (1). The lethality of inhalational anthrax is high even with antibiotic treatment and is caused by accumulation of secreted anthrax toxin (2), which consists of three proteins as follows: protective antigen (PA),² lethal factor (LF), and edema factor. PA binds to membrane receptors, forms pore complexes, and translocates LF and edema factor into the host cell (3, 4). The PA·LF complex is known as the lethal toxin, a virulence factor with pleiotropic action that facilitates establishment of the B. anthracis infection. LF is a Zn²⁺-dependent metalloprotease related to the thermolysin family that cleaves mitogen-activated protein kinase kinases (5).Although the complete mechanism by which LF causes fatal intoxication is still unclear, inhibition of LF proteolytic activity may be an efficient means of preventing anthrax lethality. A better understanding of the LF catalytic mechanism will facilitate rational design and optimization of LF inhibitors with potential clinical applicability. Recent structural (6, 7), mechanistic (8), and in vivo studies (9, 10) of LF point to a sophisticated catalytic mechanism involving accurate recognition of multiple target substrates.Here we use substrate phage display and stopped-flow fluorimetry kinetics to examine both the substrate specificity and elementary steps of substrate processing by LF. Our data allow us to construct a working model of LF-substrate binding and cleavage.     

Plants,microorganisms, and soil temperatures contribute to a decrease in methane fluxes on a drained Arctic floodplain

As surface temperatures are expected to rise in the future, ice‐rich permafrost may thaw, altering soil topography and hydrology and creating a mosaic of wet and dry soil surfaces in the Arctic. Arctic wetlands are large sources of CH₄, and investigating effects of soil hydrology on CH₄ fluxes is of great importance for predicting ecosystem feedback in response to climate change. In this study, we investigate how a decade‐long drying manipulation on an Arctic floodplain influences CH₄‐associated microorganisms, soil thermal regimes, and plant communities. Moreover, we examine how these drainage‐induced changes may then modify CH₄ fluxes in the growing and nongrowing seasons. This study shows that drainage substantially lowered the abundance of methanogens along with methanotrophic bacteria, which may have reduced CH₄ cycling. Soil temperatures of the drained areas were lower in deep, anoxic soil layers (below 30 cm), but higher in oxic topsoil layers (0–15 cm) compared to the control wet areas. This pattern of soil temperatures may have reduced the rates of methanogenesis while elevating those of CH₄ oxidation, thereby decreasing net CH₄ fluxes. The abundance of Eriophorum angustifolium, an aerenchymatous plant species, diminished significantly in the drained areas. Due to this decrease, a higher fraction of CH₄ was alternatively emitted to the atmosphere by diffusion, possibly increasing the potential for CH₄ oxidation and leading to a decrease in net CH₄ fluxes compared to a control site. Drainage lowered CH₄ fluxes by a factor of 20 during the growing season, with postdrainage changes in microbial communities, soil temperatures, and plant communities also contributing to this reduction. In contrast, we observed CH₄ emissions increased by 10% in the drained areas during the nongrowing season, although this difference was insignificant given the small magnitudes of fluxes. This study showed that long‐term drainage considerably reduced CH₄ fluxes through modified ecosystem properties.     

Ecosystem of Caspian Sea threatened by pet-traded non-indigenous crayfish

Freshwater ecosystems are particularly vulnerable to biological invasions; the aquatic animal pet trade has been recognized as a significant pathway of introductions. Crayfish are considered a model group of traded organisms with a series of highly successful species already established in the wild, having the potential to negatively influence both indigenous crayfish species (ICS) as well as alter occupied ecosystems. Eastern Europe includes the entire native ranges of indigenous Astacus leptodactylus sensu lato and A. pachypus. This region has been largely overlooked and considered relatively safe from the adverse impacts of non-indigenous crayfish species (NICS). In this study, we evaluated the crayfish pet trade in the Lower Volga Region with special emphasis on Astrakhan, the biggest city of Russian Federation in the region located just in the delta of Volga River, thus being a potential gateway of introductions to the Caspian Sea and adjacent freshwaters. The local pet trade involves 12 NICS. Considering their origin, availability, probability of establishment, invasiveness and further aspects, Procambarus fallax f. virginalis, P. clarkii and Cherax destructor are considered potentially the most problematic, including transmission of diseases like crayfish plague caused by Aphanomyces astaci or white spot syndrome virus. Taking this information as a whole, the availability of NICS with a high probability of overlapping the entire range of European ICS means that attention is warranted. Further research is needed to corroborate the abilities of NICS and their associated diseases to withstand specific conditions of the Caspian Sea as well as the adjacent Black and Azov Seas, all possessing different degrees of elevated salinity.     

Estimation of the true evolutionary distance under the fragile breakage model

Background

The ability to estimate the evolutionary distance between extant genomes plays a crucial role in many phylogenomic studies. Often such estimation is based on the parsimony assumption, implying that the distance between two genomes can be estimated as the rearrangement distance equal the minimal number of genome rearrangements required to transform one genome into the other. However, in reality the parsimony assumption may not always hold, emphasizing the need for estimation that does not rely on the rearrangement distance. The distance that accounts for the actual (rather than minimal) number of rearrangements between two genomes is often referred to as the true evolutionary distance. While there exists a method for the true evolutionary distance estimation, it however assumes that genomes can be broken by rearrangements equally likely at any position in the course of evolution. This assumption, known as the random breakage model, has recently been refuted in favor of the more rigorous fragile breakage model postulating that only certain “fragile” genomic regions are prone to rearrangements.

Results

We propose a new method for estimating the true evolutionary distance between two genomes under the fragile breakage model. We evaluate the proposed method on simulated genomes, which show its high accuracy. We further apply the proposed method for estimation of evolutionary distances within a set of five yeast genomes and a set of two fish genomes.

Conclusions

The true evolutionary distances between the five yeast genomes estimated with the proposed method reveals that some pairs of yeast genomes violate the parsimony assumption. The proposed method further demonstrates that the rearrangement distance between the two fish genomes underestimates their evolutionary distance by about 20%. These results demonstrate how drastically the two distances can differ and justify the use of true evolutionary distance in phylogenomic studies.

     

PI3K-dependent phosphorylation of Fbw7 modulates substrate degradation and activity

The Fbw7 tumor suppressor gene encodes the substrate recognition subunit of the SCF ubiquitin ligase, which targets for degradation a range of oncogenic proteins in a phosphorylation-dependent manner. Substrate phosphorylation is thought to be the main mechanism that ensures timely destruction of Fbw7 substrates. We show here that PI3K dependent phosphorylation of Fbw7 stimulates its ability to ubiquitinate and degrade its substrates. Mutation of the phosphorylation site destabilizes Fbw7 and attenuates degradation of cyclin E and Myc leading to the enhanced expression of a subset of Myc target genes. We suggest that PI3K-dependent phosphorylation of Fbw7 controls the balance between turnover of Fbw7 and its substrates to fine-tune their activity.     

Imprecision of Adaptation in Escherichia coli Chemotaxis

Adaptability is an essential property of many sensory systems, enabling maintenance of a sensitive response over a range of background stimulus levels. In bacterial chemotaxis, adaptation to the preset level of pathway activity is achieved through an integral feedback mechanism based on activity-dependent methylation of chemoreceptors. It has been argued that this architecture ensures precise and robust adaptation regardless of the ambient ligand concentration, making perfect adaptation a celebrated property of the chemotaxis system. However, possible deviations from such ideal adaptive behavior and its consequences for chemotaxis have not been explored in detail. Here we show that the chemotaxis pathway in Escherichia coli shows increasingly imprecise adaptation to higher concentrations of attractants, with a clear correlation between the time of adaptation to a step-like stimulus and the extent of imprecision. Our analysis suggests that this imprecision results from a gradual saturation of receptor methylation sites at high levels of stimulation, which prevents full recovery of the pathway activity by violating the conditions required for precise adaptation. We further use computer simulations to show that limited imprecision of adaptation has little effect on the rate of chemotactic drift of a bacterial population in gradients, but hinders precise accumulation at the peak of the gradient. Finally, we show that for two major chemoeffectors, serine and cysteine, failure of adaptation at concentrations above 1 mM might prevent bacteria from accumulating at toxic concentrations of these amino acids.     

A Conserved Isoleucine Maintains the Inactive State of Bruton's Tyrosine Kinase

Despite high level of homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamics simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state. The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest that this isoleucine side chain in the Tec family kinases acts as a “wedge” that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain.