Information-driven protein-DNA docking using HADDOCK: it is a matter of flexibility

Intrinsic flexibility of DNA has hampered the development of efficient protein−DNA docking methods. In this study we extend HADDOCK (High Ambiguity Driven DOCKing) [C. Dominguez, R. Boelens and A. M. J. J. Bonvin (2003) J. Am. Chem. Soc. 125, 1731–1737] to explicitly deal with DNA flexibility. HADDOCK uses non-structural experimental data to drive the docking during a rigid-body energy minimization, and semi-flexible and water refinement stages. The latter allow for flexibility of all DNA nucleotides and the residues of the protein at the predicted interface. We evaluated our approach on the monomeric repressor−DNA complexes formed by bacteriophage 434 Cro, the Escherichia coli Lac headpiece and bacteriophage P22 Arc. Starting from unbound proteins and canonical B-DNA we correctly predict the correct spatial disposition of the complexes and the specific conformation of the DNA in the published complexes. This information is subsequently used to generate a library of pre-bent and twisted DNA structures that served as input for a second docking round. The resulting top ranking solutions exhibit high similarity to the published complexes in terms of root mean square deviations, intermolecular contacts and DNA conformation. Our two-stage docking method is thus able to successfully predict protein−DNA complexes from unbound constituents using non-structural experimental data to drive the docking.     

Mapping hypoxia-induced bioenergetic rearrangements and metabolic signaling by 18O-assisted 31P NMR and 1H NMR spectroscopy

Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with 18O-assisted 31P NMR and 1H NMR spectroscopy. The 18O-induced isotope shift in the 31P NMR spectrum of CrP, betaADP and betaATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of 31P NMR spectra, and in both aromatic and aliphatic regions of 1H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring beta- and gamma-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. 31P and 1H NMR spectral analysis further revealed NADH and H+ scavenging by alpha-glycerophosphate dehydrogenase (alphaGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of alpha-glycerophosphate and nucleoside 5'-monophosphates, through alphaGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the 31P NMR spectrum. Thus, 18O-assisted 31P NMR combined with 1H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress.     

The effect of ozone exposure on the release of eicosanoids in guinea-pig BAL fluid in relation to cellular damage and inflammation

The observed effects after ozone exposure strongly depend on ozone concentration and exposure time. We hypothesized that depending on the O3 exposure protocol, mainly either an oxidant damage or an inflammation will determine the O3 toxicity. We compared two different ozone exposure protocols: an acute exposure (3 ppm 2 h) for studying the oxidant damage and an exposure (1 ppm 12 h) where an inflammatory component is also probably involved. We measured LDH activity and protein and albumin exudation as markers for cellular damage. After the acute exposure an increase in LDH activity was measured and after exposure to 1 ppm ozone for 12 h the exudation of protein and albumin was also enhanced. The histological examinations showed a neutrophilic inflammatory response only after exposure to 1 ppm ozone for 12 h. The acute exposure protocol resulted in an increased release of PGE2, PGD2, PGF2alpha and 6-ketoPGF1alpha whereas exposure to 1 ppm ozone for 12 h led to an additional release of LTB4. No effects were measured on the release of TxB2 and LTC4/D4/E4. These changed amounts of eicosanoids will probably contribute to the ozone-induced lung function changes.     

Defective metabolic signaling in adenylate kinase AK1 gene knock-out hearts compromises post-ischemic coronary reflow

Matching blood flow to myocardial energy demand is vital for heart performance and recovery following ischemia. The molecular mechanisms responsible for transduction of myocardial energetic signals into reactive vasodilatation are, however, elusive. Adenylate kinase, associated with AMP signaling, is a sensitive reporter of the cellular energy state, yet the contribution of this phosphotransfer system in coupling myocardial metabolism with coronary flow has not been explored. Here, knock out of the major adenylate kinase isoform, AK1, disrupted the synchrony between inorganic phosphate P(i) turnover at ATP-consuming sites and gamma-ATP exchange at ATP synthesis sites, as revealed by (18)O-assisted (31)P NMR. This reduced energetic signal communication in the post-ischemic heart. AK1 gene deletion blunted vascular adenylate kinase phosphotransfer, compromised the contractility-coronary flow relationship, and precipitated inadequate coronary reflow following ischemia-reperfusion. Deficit in adenylate kinase activity abrogated AMP signal generation and reduced the vascular adenylate kinase/creatine kinase activity ratio essential for the response of metabolic sensors. The sarcolemma-associated splice variant AK1beta facilitated adenosine production, a function lost in the absence of adenylate kinase activity. Adenosine treatment bypassed AK1 deficiency and restored post-ischemic flow to wild-type levels, achieving phenotype rescue. AK1 phosphotransfer thus transduces stress signals into adequate vascular response, providing linkage between cell bioenergetics and coronary flow.     

Various strategies of using residual dipolar couplings in NMR-driven protein docking: application to Lys48-linked di-ubiquitin and validation against 15N-relaxation data

When classical, Nuclear Overhauser Effect (NOE)-based approaches fail, it is possible, given high-resolution structures of the free molecules, to model the structure of a complex in solution based solely on chemical shift perturbation (CSP) data in combination with orientational restraints from residual dipolar couplings (RDCs) when available. RDCs can be incorporated into the docking following various strategies: as direct restraints and/or as intermolecular intervector projection angle restraints (Meiler et al., J Biomol NMR 2000;16:245-252). The advantage of the latter for docking is that they directly define the relative orientation of the molecules. A combined protocol in which RDCs are first introduced as intervector projection angle restraints and at a later stage as direct restraints is shown here to give the best performance. This approach, implemented in our information-driven docking approach HADDOCK (Dominguez et al., J Am Chem Soc 2003;125:1731-1737), is used to determine the solution structure of the Lys48-linked di-ubiquitin, for which chemical shift mapping, RDCs, and (15)N-relaxation data have been previously obtained (Varadan et al., J Mol Biol 2002;324:637-647). The resulting structures, derived from CSP and RDC data, are cross-validated using (15)N-relaxation data. The solution structure differs from the crystal structure by a 20 degrees rotation of the two ubiquitin units relative to each other.     

Bayesian Markov Random Field Analysis for Protein Function Prediction Based on Network Data

Inference of protein functions is one of the most important aims of modern biology. To fully exploit the large volumes of genomic data typically produced in modern-day genomic experiments, automated computational methods for protein function prediction are urgently needed. Established methods use sequence or structure similarity to infer functions but those types of data do not suffice to determine the biological context in which proteins act. Current high-throughput biological experiments produce large amounts of data on the interactions between proteins. Such data can be used to infer interaction networks and to predict the biological process that the protein is involved in. Here, we develop a probabilistic approach for protein function prediction using network data, such as protein-protein interaction measurements. We take a Bayesian approach to an existing Markov Random Field method by performing simultaneous estimation of the model parameters and prediction of protein functions. We use an adaptive Markov Chain Monte Carlo algorithm that leads to more accurate parameter estimates and consequently to improved prediction performance compared to the standard Markov Random Fields method. We tested our method using a high quality S.cereviciae validation network with 1622 proteins against 90 Gene Ontology terms of different levels of abstraction. Compared to three other protein function prediction methods, our approach shows very good prediction performance. Our method can be directly applied to protein-protein interaction or coexpression networks, but also can be extended to use multiple data sources. We apply our method to physical protein interaction data from S. cerevisiae and provide novel predictions, using 340 Gene Ontology terms, for 1170 unannotated proteins and we evaluate the predictions using the available literature.     

Phase 2 study of canfosfamide in combination with pegylated liposomal doxorubicin in platinum and paclitaxel refractory or resistant epithelial ovarian cancer

We have reviewed the pivotal presentations related to gastrointestinal malignancies from 2009 annual meeting of the American Society of Clinical Oncology with the theme of "personalizing cancer care". We have discussed the scientific findings and the impact on practice guidelines and ongoing clinical trials. Adding trastuzumab to chemotherapy improved the survival of patients with advanced gastric cancer overexpressing human epidermal growth factor receptor 2. Gemcitabine plus cisplatin has become a new standard for first-line treatment of advanced biliary cancer. Octreotide LAR significantly lengthened median time to tumor progression compared with placebo in patients with metastatic neuroendocrine tumors of the midgut. Addition of oxaliplatin to fluoropyrimidines for preoperative chemoradiotherapy in patients with stage II or III rectal cancer did not improve local tumor response but increased toxicities. Bevacizumab did not provide additional benefit to chemotherapy in adjuvant chemotherapy for stage II or III colon cancer. In patients with resected stage II colon cancer, recurrence score estimated by multigene RT-PCR assay has been shown to provide additional risk stratification. In stage IV colorectal cancer, data have supported the routine use of prophylactic skin treatment in patients receiving antibody against epidermal growth factor receptor, and the use of upfront chemotherapy as initial management in patients with synchronous metastasis without obstruction or bleeding from the primary site.     

Length composition and abundance of eel larvae,Anguilla anguilla (anguilliformes: Anguillidae), in the iberian basin (northeastern Atlantic) during July–September 1984

376 leptocephali ofAnguilla anguilla (L., 1758) from the Iberian Basin were analysed. The observed horizontal trends of abundance and particularly the mean sizes contradict the expectations based on the hypothesis of larvae distribution exclusively by drift with Gulf and North Atlantic Currents, and support the hypothesis of an active larval migration also south of the Azores.