Vegetation feedbacks of nutrient addition lead to a weaker carbon sink in an ombrotrophic bog

To study vegetation feedbacks of nutrient addition on carbon sequestration capacity, we investigated vegetation and ecosystem CO₂ exchange at Mer Bleue Bog, Canada in plots that had been fertilized with nitrogen (N) or with N plus phosphorus (P) and potassium (K) for 7–12 years. Gross photosynthesis, ecosystem respiration, and net CO₂ exchange were measured weekly during May–September 2011 using climate‐controlled chambers. A substrate‐induced respiration technique was used to determine the functional ability of the microbial community. The highest N and NPK additions were associated with 40% less net CO₂ uptake than the control. In the NPK additions, a diminished C sink potential was due to a 20–30% increase in ecosystem respiration, while gross photosynthesis rates did not change as greater vascular plant biomass compensated for the decrease in Sphagnum mosses. In the highest N‐only treatment, small reductions in gross photosynthesis and no change in ecosystem respiration led to the reduced C sink. Substrate‐induced microbial respiration was significantly higher in all levels of NPK additions compared with control. The temperature sensitivity of respiration in the plots was lower with increasing cumulative N load, suggesting more labile sources of respired CO₂. The weaker C sink potential could be explained by changes in nutrient availability, higher woody : foliar ratio, moss loss, and enhanced decomposition. Stronger responses to NPK fertilization than to N‐only fertilization for both shrub biomass production and decomposition suggest that the bog ecosystem is N‐P/K colimited rather than N‐limited. Negative effects of further N‐only deposition were indicated by delayed spring CO₂ uptake. In contrast to forests, increased wood formation and surface litter accumulation in bogs seem to reduce the C sink potential owing to the loss of peat‐forming Sphagnum.     

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.     

Evaluation of structural features in fungal cytochromes P450 predicted to rule catalytic diversification

Fungi belong to the large kingdom of lower eukaryotic organisms encompassing yeasts along with filamentous and dimorphic members. Microbial P450 enzymes have contributed to exploration of and adaptation to diverse ecological niches such as conversion of lipophilic compounds to more hydrophilic derivatives or degradation of a vast array of environmental toxicants. To better understand diversification of the catalytic behavior of fungal P450s, detailed insight into the molecular machinery steering oxidative attack on the distinctly structured endogenous and xenobiotic substrates is of preeminent interest. Based on a general, CYP102A1-related template the bulk of predicted substrate/inhibitor-binding determinants were shown to cluster near the distal heme face within the six known substrate recognition sites (SRSs) made up by the α-helical B′/F/G/I tetrad, the B′–C interhelical loop and strands of the β6-sheet, population density being highest in the structurally flexible SRS-1 and SRS-4 domains, showing a low degree of conservation. Reactivity toward ligands favorably coincides with the lipophilicity/hydrophilicity profile and bulkiness of critical amino acids acting as selective filters. Some decisive elements may also serve in maintenance of catalytic competence via their action as gatekeepers directing substrate access/positioning or stabilizers of the heme environment enabling dioxygen activation. Non-SRS residues seem to control spin state equilibria and attract redox partners by electrostatic forces. Of note, the inhibitory potency of azole-type fungicides is likely to arise from perturbation of the complex interplay of the mechanistic principles addressed above. Knowledge-supported exploitation of the topological data will be helpful in the manufacture of commodity/specialty chemicals as well as therapeutic agents. Also, engineered fungal P450s may be used to improve pollutant-specific bioremediation of contaminated soils.     

Overcoming resistance to rapalogs in gliomas by combinatory therapies

Glioblastoma is the most common and aggressive brain tumor type, with a mean patient survival of approximately 1 year. Many previous analyses of the glioma kinome have identified key deregulated pathways that converge and activate mammalian target of rapamycin (mTOR). Following the identification and characterization of mTOR-promoting activity in gliomagenesis, data from preclinical studies suggested the targeting of mTOR by rapamycin or its analogs (rapalogs) as a promising therapeutic approach. However, clinical trials with rapalogs have shown very limited efficacy on glioma due to the development of resistance mechanisms. Analysis of rapalog-insensitive glioma cells has revealed increased activity of growth and survival pathways compensating for mTOR inhibition by rapalogs that are suitable for therapeutic intervention. In addition, recently developed mTOR inhibitors show high anti-glioma activity. In this review, we recapitulate the regulation of mTOR signaling and its involvement in gliomagenesis, discuss mechanisms resulting in resistance to rapalogs, and speculate on strategies to overcome resistance. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation

In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksb is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation.     

π–π Stacking mediated drug–drug interactions in human CYP2E1

Because of having many low molecular mass substrates, CYP2E1 is of particular interests to the pharmaceutical industry. Many evidences showed that this enzyme can adopt multiple substrates to significantly reduce the oxidation rate of the substrates. The detailed mechanism for this observation is still unclear. In the current study, we employed GPU‐accelerated molecular dynamics simulations to study the multiple‐binding mode of human CYP2E1, with an aim of offering a mechanistic explanation for the unexplained multiple‐substrate binding. Our results showed that Thr303 and Phe478 were key factors for the substrate recognition and multiple‐substrate binding. The former can form a significant hydrogen bond to recognize and position the substrate in the productive binding orientation in the active site. The latter acted as a mediator for the substrate communications via π–π stacking interactions. In the multiple‐binding mode, the aforementioned π–π stacking interactions formed by the aromatic rings of both substrates and Phe478 drove the first substrate far away from the catalytic center, orienting in an additional binding position and going against the substrate metabolism. All these findings could give atomic insights into the detailed mechanism for the multiple‐substrate binding in human CYP2E1, providing useful information for the drug metabolism mechanism and personalized use of clinical drugs. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

LabCaS: Labeling calpain substrate cleavage sites from amino acid sequence using conditional random fields

The calpain family of Ca²⁺‐dependent cysteine proteases plays a vital role in many important biological processes which is closely related with a variety of pathological states. Activated calpains selectively cleave relevant substrates at specific cleavage sites, yielding multiple fragments that can have different functions from the intact substrate protein. Until now, our knowledge about the calpain functions and their substrate cleavage mechanisms are limited because the experimental determination and validation on calpain binding are usually laborious and expensive. In this work, we aim to develop a new computational approach (LabCaS) for accurate prediction of the calpain substrate cleavage sites from amino acid sequences. To overcome the imbalance of negative and positive samples in the machine‐learning training which have been suffered by most of the former approaches when splitting sequences into short peptides, we designed a conditional random field algorithm that can label the potential cleavage sites directly from the entire sequences. By integrating the multiple amino acid features and those derived from sequences, LabCaS achieves an accurate recognition of the cleave sites for most calpain proteins. In a jackknife test on a set of 129 benchmark proteins, LabCaS generates an AUC score 0.862. The LabCaS program is freely available at: http://www.csbio.sjtu.edu.cn/bioinf/LabCaS . Proteins 2013. © 2012 Wiley Periodicals, Inc.