Experimental detection of short regulatory motifs in eukaryotic proteins: tips for good practice as well as for bad

It has become clear in outline though not yet in detail how cellular regulatory and signalling systems are constructed. The essential machines are protein complexes that effect regulatory decisions by undergoing internal changes of state. Subcomponents of these cellular complexes are assembled into molecular switches. Many of these switches employ one or more short peptide motifs as toggles that can move between one or more sites within the switch system, the simplest being on-off switches. Paradoxically, these motif modules (termed short linear motifs or SLiMs) are both hugely abundant but difficult to research. So despite the many successes in identifying short regulatory protein motifs, it is thought that only the “tip of the iceberg” has been exposed. Experimental and bioinformatic motif discovery remain challenging and error prone. The advice presented in this article is aimed at helping researchers to uncover genuine protein motifs, whilst avoiding the pitfalls that lead to reports of false discovery.     

Classification with High‐Dimensional Genetic Data: Assigning Patients and Genetic Features to Known Classes

A major task in the statistical analysis of genetic data such as gene expressions and single nucleotide polymorphisms (SNPs) is to predict whether a patient has a certain disease, or from which of several known subtypes of a disease a patient suffers. A large number of discrimination methods have been proposed in the literature and have been applied to genetic data to tackle this task. In this paper, we give an overview on the most popular of these procedures in the analysis of genetic data. Moreover, we describe how these methods for supervised classification can be combined with variable selection approaches to reduce the number of genetic features from several thousands to as few as possible to form a concise classification rule. Finally, we show how the resulting statistical models can be validated. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Identification of ectonucleotidases CD39 and CD73 in innate protection during acute lung injury

Acute lung injury (ALI), such as that which occurs with mechanical ventilation, contributes to morbidity and mortality of critical illness. Nonetheless, in many instances, ALI resolves spontaneously through unknown mechanisms. Therefore, we hypothesized the presence of innate adaptive pathways to protect the lungs during mechanical ventilation. In this study, we used ventilator-induced lung injury as a model to identify endogenous mechanisms of lung protection. Initial in vitro studies revealed that supernatants from stretch-induced injury contained a stable factor which diminished endothelial leakage. This factor was subsequently identified as adenosine. Additional studies in vivo revealed prominent increases in pulmonary adenosine levels with mechanical ventilation. Because ectoapyrase (CD39) and ecto-5'-nucleotidase (CD73) are rate limiting for extracellular adenosine generation, we examined their contribution to ALI. In fact, both pulmonary CD39 and CD73 are induced by mechanical ventilation. Moreover, we observed pressure- and time-dependent increases in pulmonary edema and inflammation in ventilated cd39(-/-) mice. Similarly, pharmacological inhibition or targeted gene deletion of cd73 was associated with increased symptom severity of ventilator-induced ALI. Reconstitution of cd39(-/-) or cd73(-/-) mice with soluble apyrase or 5'-nucleotidase, respectively, reversed such increases. In addition, ALI was significantly attenuated and survival improved after i.p. treatment of wild-type mice with soluble apyrase or 5'-nucleotidase. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in lung protection and suggest treatment with their soluble compounds as a therapeutic strategy for noninfectious ALI.     

Structural and thermodynamic study on aldose reductase: nitro-substituted inhibitors with strong enthalpic binding contribution

To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the development of aldose reductase inhibitors (ARIs) has been established as a promising therapeutic concept. In order to identify novel lead compounds, a virtual screening (VS) was performed successfully suggesting carboxylate-type inhibitors of sub-micromolar to micromolar affinity. Here, we combine a structural characterization of the binding modes observed by X-ray crystallography with isothermal titration calorimetry (ITC) measurements providing insights into the driving forces of inhibitor binding, particularly of the first leads from VS. Characteristic features of this novel inhibitor type include a carboxylate head group connected via an alkyl spacer to a heteroaromatic moiety, which is linked to a further nitro-substituted aromatic portion. The crystal structures of two enzyme-inhibitor complexes have been determined at resolutions of 1.43 A and 1.55 A. Surprisingly, the carboxylic group of the most potent VS lead occupies the catalytic pocket differently compared to the interaction geometry observed in almost all other crystal structures with structurally related ligands and obtained under similar conditions, as an interstitial water molecule is picked up upon ligand binding. The nitro-aromatic moiety of both leads occupies the specificity pocket of the enzyme, however, adopting a different geometry compared to the docking prediction: unexpectedly, the nitro group binds to the bottom of the specificity pocket and provokes remarkable induced-fit adaptations. A peptide group located at the active site orients in such a way that H-bond formation to one nitro group oxygen atom is enabled, whereas a neighbouring tyrosine side-chain performs a slight rotation off from the binding cavity to accommodate the nitro group. Identically constituted ligands, lacking this nitro group, exhibit an affinity drop of one order of magnitude. In addition, thermodynamic data suggest a strongly favourable contribution to binding enthalpy in case the inhibitor is equipped with a nitro group at the corresponding position. To further investigate this phenomenon, we determined crystal structures and thermodynamic data of two similarly constituted IDD-type inhibitors addressing the specificity pocket with either a nitro or halogen-substituted aromatic moiety. As these data suggest, the nitro group provokes the enthalpic contribution, in addition to the H-bond mentioned above, by accepting two "non-classical" H-bonds donated by the aromatic tyrosine side-chain. In summary, this study provides the platform for further structure-guided design hypotheses of novel drug candidates with higher affinity and selectivity.     

CD101 surface expression discriminates potency among murine FoxP3+ regulatory T cells

CD4+CD25+FoxP3+ regulatory T cells (Treg) have been shown to be protective in animal models of autoimmunity and acute graft-vs-host disease. However, owing to the functional heterogeneity among CD4+CD25+ T cells, surface markers expressed selectively on functionally active Treg would be useful for purposes of identifying and isolating such cells. We generated a rabbit mAb against murine CD101, a transmembrane glycoprotein involved in T cell activation. Among freshly isolated T cells, CD101 was detected on 25-30% of CD4+CD25+ Treg and approximately 20% of conventional memory T cells. CD101(high) Treg displayed greater in vitro suppression of alloantigen-driven T cell proliferation as compared with CD101(low) Treg. In a model of graft-vs-host disease induced by allogeneic bone marrow transplantation in vivo bioluminescence imaging demonstrated reduced expansion of donor-derived luciferase-labeled conventional T cells in mice treated with CD101(high) Treg, compared with CD101(low) Treg. Moreover, treatment with CD101(high) Treg resulted in improved survival, reduced proinflammatory cytokine levels and reduced end organ damage. Among the CD101(high) Treg all of the in vivo suppressor activity was contained within the CD62L(high) subpopulation. We conclude that CD101 expression distinguishes murine Treg with potent suppressor activity.     

Spiralian phylogenomics supports the resurrection of Bryozoa comprising Ectoprocta and Entoprocta

Phylogenetic analyses based on 79 ribosomal proteins of 38 metazoans, partly derived from 6 new expressed sequence tag projects for Ectoprocta, Entoprocta, Sipuncula, Annelida, and Acanthocephala, indicate the monophyly of Bryozoa comprising Ectoprocta and Entoprocta, 2 taxa that have been separated for more than a century based on seemingly profound morphological differences. Our results also show that bryozoans are more closely related to Neotrochozoa, including molluscs and annelids, than to Syndermata, the latter comprising Rotifera and Acanthocephala. Furthermore, we find evidence for the position of Sipuncula within Annelida. These findings suggest that classical developmental and morphological key characters such as cleavage pattern, coelomic cavities, gut architecture, and body segmentation are subject to greater evolutionary plasticity than traditionally assumed.     

Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo

We have used Salmonella enterica serovar Typhimurium (S. typhimurium) which are able to colonize tumours besides spleen and liver. Bacteria were equipped with constructs encoding green fluorescent protein or luciferase as reporters under control of the promoter PBAD that is inducible with L-arabinose. Reporter genes could be induced in culture but also when the bacteria resided within the mouse macrophages J774A.1. More important, strong expression of reporters by the bacteria could be detected in mice after administration of L-arabinose. This was especially pronounced in bacteria colonizing tumours. Histology demonstrated that the bacteria had accumulated in and close to necrotic areas of tumours. Bacterial gene induction was observed in both regions. PBAD is tightly controlled also in vivo because gene E of bacteriophage PhiX174 could be introduced as inducible suicide gene. The possibility to deliberately induce genes in bacterial carriers within the host should render them extremely powerful tools for tumour therapy.     

Capturing cyclic nucleotides in action: snapshots from crystallographic studies

Fifty years ago, cyclic AMP was discovered as a second messenger of hormone action, heralding the age of signal transduction. Many cellular processes were found to be regulated by cAMP and the related cyclic GMP. Cyclic nucleotides function by binding to and activating their effectors - protein kinase A, protein kinase G, cyclic-nucleotide-regulated ion channels and the guanine nucleotide-exchange factor Epac. Recent structural insights have now made it possible to propose a general structural mechanism for how cyclic nucleotides regulate these proteins.     

A replica exchange Monte Carlo algorithm for protein folding in the HP model

Background  

The ab initio protein folding problem consists of predicting protein tertiary structure from a given amino acid sequence by minimizing an energy function; it is one of the most important and challenging problems in biochemistry, molecular biology and biophysics. The ab initio protein folding problem is computationally challenging and has been shown to be -hard even when conformations are restricted to a lattice. In this work, we implement and evaluate the replica exchange Monte Carlo (REMC) method, which has already been applied very successfully to more complex protein models and other optimization problems with complex energy landscapes, in combination with the highly effective pull move neighbourhood in two widely studied Hydrophobic Polar (HP) lattice models.