Global remodelling of cellular microenvironment due to loss of collagen VII

The mammalian cellular microenvironment is shaped by soluble factors and structural components, the extracellular matrix, providing physical support, regulating adhesion and signalling. A global, quantitative mass spectrometry strategy, combined with bioinformatics data processing, was developed to assess proteome differences in the microenvironment of primary human fibroblasts. We studied secreted proteins of fibroblasts from normal and pathologically altered skin and their post‐translational modifications. The influence of collagen VII, an important structural component, which is lost in genetic skin fragility, was used as model. Loss of collagen VII had a global impact on the cellular microenvironment and was associated with proteome alterations highly relevant for disease pathogenesis including decrease in basement membrane components, increase in dermal matrix proteins, TGF‐β and metalloproteases, but not higher protease activity. The definition of the proteome of fibroblast microenvironment and its plasticity in health and disease identified novel disease mechanisms and potential targets of intervention.     

Design and synthesis of xanthine analogues as potent and selective PDE5 inhibitors

We have discovered potent and selective xanthine PDE5 inhibitors. Compound 25 (PDE5 IC(50)=0.6 nM, PDE6/PDE5=101) demonstrated similar functional efficacy and PK profile to Sildenafil (PDE5 IC(50)=3.5 nM, PDE6/PDE5=7).     

Skeletal muscle glucose uptake following overload-induced hypertrophy.

While endurance exercise training has been shown to enhance insulin action in skeletal muscle, the effects of high resistance strength training are less clear. The purpose of this study was to determine the rate of glucose uptake in skeletal muscle in which compensatory hypertrophy was induced by synergist muscle ablation. Basal and insulin mediated [3H] 2-deoxyglucose uptake were measured in soleus and EDL muscles using the perfused rat hindquarter preparation. Neither basal nor insulin mediated glucose uptake, when expressed per gram muscle, were enhanced in hypertrophied soleus muscles compared with control muscles, despite a twofold increase in mass (P less than 0.01). In the EDL, muscle mass increased 60% with synergist ablation (P less than 0.01), however insulin mediated glucose uptake was not different from that of control muscles. The basal rate of glucose uptake in hypertrophied EDL muscles was increased twofold over that of control muscles (P less than 0.05), possibly due to changes in neural input and/or loading. These results suggest that the stimulus for development of increased muscle mass is different from that for metabolic adaptations.     

SAR development of polycyclic guanine derivatives targeted to the discovery of a selective PDE5 inhibitor for treatment of erectile dysfunction

Development of structure-activity relationship of cyclic guanines I lead us to discovery of a potent and selective series of phosphodiesterase 5 inhibitors 52-59 (IC50=1.3-11.0 nM, PDE6/5=116-600).     

Probabilistic cross-link analysis and experiment planning for high-throughput elucidation of protein structure

Emerging high-throughput techniques for the characterization of protein and protein-complex structures yield noisy data with sparse information content, placing a significant burden on computation to properly interpret the experimental data. One such technique uses cross-linking (chemical or by cysteine oxidation) to confirm or select among proposed structural models (e.g., from fold recognition, ab initio prediction, or docking) by testing the consistency between cross-linking data and model geometry. This paper develops a probabilistic framework for analyzing the information content in cross-linking experiments, accounting for anticipated experimental error. This framework supports a mechanism for planning experiments to optimize the information gained. We evaluate potential experiment plans using explicit trade-offs among key properties of practical importance: discriminability, coverage, balance, ambiguity, and cost. We devise a greedy algorithm that considers those properties and, from a large number of combinatorial possibilities, rapidly selects sets of experiments expected to discriminate pairs of models efficiently. In an application to residue-specific chemical cross-linking, we demonstrate the ability of our approach to plan experiments effectively involving combinations of cross-linkers and introduced mutations. We also describe an experiment plan for the bacteriophage lambda Tfa chaperone protein in which we plan dicysteine mutants for discriminating threading models by disulfide formation. Preliminary results from a subset of the planned experiments are consistent and demonstrate the practicality of planning. Our methods provide the experimenter with a valuable tool (available from the authors) for understanding and optimizing cross-linking experiments.     

Phylogenomic identification of five new human homologs of the DNA repair enzyme AlkB

Background  

Combination of biochemical and bioinformatic analyses led to the discovery of oxidative demethylation - a novel DNA repair mechanism catalyzed by theEscherichia coliAlkB protein and its two human homologs, hABH2 and hABH3. This discovery was based on the prediction made by Aravind and Koonin that AlkB is a member of the 2OG-Fe²⁺oxygenase superfamily.